Search Thermo Fisher Scientific
Search Thermo Fisher Scientific
The CloneMiner cDNA Library Construction Kit is designed to construct high-quality cDNA libraries without the use of traditional restriction enzyme cloning methods. This novel technology combines the performance of SuperScript II Reverse Transcriptase with the Gateway Technology.
Single-stranded mRNA is converted into double stranded cDNA containing attB sequences on each end. Through site-specific recombination, attB-flanked cDNA is cloned directly into an attP-containing donor vector without the use of restriction digestion or ligation.
The resulting Gateway entry cDNA library can be screened with a probe to identify a specific entry clone. This clone can be transferred into the Gateway destination vector of choice for gene expression and functional analysis. Alternatively, the entire entry cDNA library can be shuttled into a Gateway destination vector to generate an expression library.
Features of the CloneMiner cDNA Library Construction Kit include:
Advantages of the CloneMiner cDNA Library Construction Kit
Using CloneMiner cDNA Library Construction Kit offers the following advantages:
Experimental Summary
The following diagram summarizes the cDNA synthesis process of the CloneMiner cDNA Library Construction Kit.
The Gateway Technology
Gateway is a universal cloning technology based on the site-specific recombination properties of bacteriophage lambda (Landy, 1989). The Gateway Technology provides a rapid and highly efficient way to move DNA sequences into multiple vector systems for functional analysis and protein expression. For more information on the Gateway Technology, see next section.
Introduction
There are several ways to construct your cDNA library using the CloneMiner cDNA Library Construction Kit. You will need to decide between:
We recommend radiolabeling your cDNA and size fractionating your cDNA by column chromatography. This section provides information to help you choose the library construction method that best suits your needs.
Radiolabeling vs. Non-Radiolabeling
The table below outlines the advantages and disadvantages of the radiolabeling and non-radiolabeling methods. Use this information to choose one method to construct your cDNA library.
Radiolabeling Method | Non-Radiolabeling Method | |
Analyzing First Strand Synthesis |
Direct measure of cDNA yield and overall quality of the first strand
|
No knowledge of cDNA yield or quality until the library is constructed
|
Determining cDNA Yields for Cloning |
Reliable quantitative method using scintillation counter
|
Qualitative, subjective method using agarose plate spotting assay
|
Sensitivity of cDNA Detection |
Very sensitive to a wide range of cDNA amounts using scintillation counter
|
Sensitive in detecting 1-10 ng of cDNA per spot (see
Performing the Plate Spotting Assay).
Limited resolution for cDNA yields greater than 10 ng per spot (see
Performing the Plate Spotting Assay).
|
Experimental Time |
Time consuming filter washes, counting samples, performing calculations
|
DNA standards and plates for the plate spotting assay can be prepared in advance for several experiments, limited calculations
|
Preparation |
Requires extensive preparation of reagents, equipment, and work area
|
Requires minimal preparation of DNA standards and agarose plates for the plate spotting assay
|
Lab Environment |
Need to work in designated areas, dispose of radioactive waste, monitor work area, follow radioactive safety regulations
|
Regular lab environment with no radioactive hazards or radioactive safety regulations
|
Be sure to read the section entitled Advance Preparation, to prepare any necessary reagents required for your method of choice. If you will be using the radiolabeling method, also read the section entitled Working with Radioactive Materials. If you will be using the non-radiolabeling method, we recommend that you read the section entitled Performing the Plate Spotting Assay, before beginning.
Choosing a Size Fractionation Method
Size fractionation generates cDNA that is free of adapters and other low molecular weight DNA. Although we recommend size fractionating your cDNA by column chromatography, you may also size fractionate your cDNA by gel electrophoresis. Either method can be used with radiolabeled or non-radiolabeled cDNA. Refer to the guidelines outlined below and choose the method that best suits your needs.
Column Chromatography
Column chromatography is commonly used to size fractionate cDNA. Use the column chromatography method to generate a cDNA library with an average cDNA insert size of approximately 1.5 kb (if you start with high-quality mRNA).
Protocols to size fractionate radiolabeled or non-radiolabeled cDNA by column chromatography are provided in this protocol.
Gel Electrophoresis
Use the gel electrophoresis method to generate a cDNA library with a larger average insert size (>2.0 kb) or to select cDNA of a particular size. Protocols to size fractionate radiolabeled or non-radiolabeled cDNA by gel electrophoresis are provided in the CloneMiner cDNA Construction Kit Web Appendix. Because you will need to have additional reagents on hand, we recommend reading the Web Appendix before beginning. This manual is available from our Web site (www.thermofisher.com) or by contacting Technical Service.
The CloneMiner cDNA Library Construction Kit is designed to help you construct a cDNA library without the use of traditional restriction enzyme cloning methods. Use of this kit is geared towards those users who have some familiarity with cDNA library construction. We highly recommend that users possess a working knowledge of mRNA isolation and library construction techniques before using this kit.
For more information about these topics, refer to the following published reviews:
Introduction
Read the following section if you will be constructing your cDNA library using a radiolabeled isotope. This section provides general guidelines and safety tips for working with radioactive material. For more information and specific requirements, contact the safety department of your institution.
Use extreme caution when working with radioactive material. Follow all federal and state regulations regarding radiation safety. For general guidelines when working with radioactive material, see below.
General Guidelines
Follow these general guidelines when working with radioactive material.
Any material in contact with a radioactive isotope must be disposed of properly. This will include any reagents that are discarded during the cDNA library synthesis procedure (e.g. phenol/chloroform extraction, ethanol precipitation, cDNA size fractionation). Contact your safety department for regulations regarding radioactive waste disposal.
Introduction
The CloneMiner cDNA Library Construction Kit is designed to produce an entry library from your starting mRNA within three days. It will take an additional two days to determine the titer and quality of the cDNA library. Note that this protocol is organized according to the recommended timeline below. If you will not be following this timeline, be sure to plan ahead for convenient stopping points (see below for more information).
Recommended Timeline
If you are performing the radiolabeling method, we recommend that you follow the timeline outlined above. Radiochemical effects induced by 32P decay in the cDNA can reduce transformation efficiencies over time.
Optional Stopping Points
If you cannot follow the recommended timeline, you may stop the procedure during any ethanol precipitation step. These steps occur during second strand synthesis and size fractionation and are noted as optional stopping points. When stopping at these points, always store the cDNA as the uncentrifuged ethanol precipitate at -20°C to maximize cDNA stability.
Introduction
The experimental steps necessary to synthesize attB-flanked cDNA and to generate an entry library are outlined below. Once you have isolated your mRNA, you will need a minimum of 3 days to construct a cDNA library.
Day | Step | Action |
1 |
1
|
Synthesize the first strand of cDNA from your isolated mRNA using the Biotin-
attB2-Oligo(dT) Primer and SuperScript
™ II RT.
|
2
|
Synthesize the second strand of cDNA using the first strand cDNA as a template.
| |
3
|
Analyze the first strand reaction for cDNA yield and percent incorporation of [a-
32P]dCTP.
| |
4
|
Ligate the
attB1 adapter to the 5'
end of your cDNA.
| |
2 |
1
|
Size fractionate the cDNA by column chromatography to remove excess primers, adapters, and small cDNA.
|
2
|
Perform the BP recombination reaction between the
attB-flanked cDNA and pDONR
™222.
| |
3 |
1
|
Transform the BP reactions into ElectroMAX
™ DH10B
™ T1 Phage Resistant cells. Add freezing media to transformed cells to get final cDNA library.
|
2
|
Perform the plating assay to determine the cDNA library titer.
| |
4-5 |
1
|
Calculate the cDNA library titer using the results from the plating assay.
|
2
|
Inoculate 24 positive transformants from the plating assay. Determine average insert size and percent recombinants by restriction analysis.
| |
3
|
Sequence entry clones to verify presence of cDNA insert, if desired.
|
Introduction
You will need to isolate high-quality mRNA using a method of choice prior to using this kit. Follow the guidelines provided below to avoid RNase contamination.
Aerosol-resistant pipette tips are recommended for all procedures. See below for general recommendations for handling mRNA.
General Handling of mRNA
When working with mRNA:
You may use RNase Away Reagent, a non-toxic solution available from Invitrogen, to remove RNase contamination from surfaces. For further information on controlling RNase contamination, see Current Protocols in Molecular Biology (Ausubel et al., 1994) or Molecular Cloning: A Laboratory Manual (Sambrook et al., 1989).
mRNA Isolation
mRNA can be isolated from tissue, cells, or total RNA using the method of choice. We recommend isolating mRNA using the Micro-FastTrack 2.0 or FastTrack 2.0 mRNA Isolation Kits available from Invitrogen. Generally, 1 to 5 µg of mRNA will be sufficient to construct a cDNA library containing 106 to 107 primary clones in E. coli. Resuspend isolated mRNA in DEPC-treated water and check the quality of your preparation. Store your mRNA preparation at -80°C. We recommend aliquoting your mRNA into multiple tubes to reduce the number of freeze/thaw cycles.
It is very important to use the highest quality mRNA possible to ensure success. Check the integrity and purity of your mRNA before starting.
Checking the Total RNA Quality
To check total RNA integrity, analyze 1 µg of your RNA by agarose/ethidium bromide gel electrophoresis. You should see the following on a denaturing agarose gel:
Checking the mRNA Quality
mRNA will appear as a smear from 0.5 to 12 kb. rRNA bands may still be faintly visible. If you do not detect a smear or if the smear is running significantly smaller than 12 kb, you will need to repeat the RNA isolation. Be sure to follow the recommendations listed on the previous page to prevent RNase contamination.
Introduction
Some of the reagents and materials required to use the CloneMiner cDNA Library Construction Kit are not supplied with the kit and may not be common lab stock. Refer to the lists below to help you prepare or acquire these materials in advance.
Refer to the section entitled Before Starting at the beginning of each procedure for a complete list of required reagents.
Materials Required for the Radiolabeling Method
You should have the following materials on hand before performing the radiolabeling method:
Materials Required for the Non-Radiolabeling Method
You should have the following on hand before performing the non-radiolabeling method.
Number of Reactions
This kit provides enough reagents to construct five cDNA libraries. While some reagents are supplied in excess, you may need additional reagents and materials if you wish to perform more than 5 reactions. You may also need additional electrocompetent E. coli cells if you will be performing control reactions (2.3 kb RNA control, pEXP7-tet control, BP negative control, and pUC 19 transformation control) each time you construct a cDNA library.
Synthesizing the First Strand
Introduction
This section provides detailed guidelines for synthesizing the first strand of cDNA from your isolated mRNA. The reaction conditions for first strand synthesis catalyzed by SuperScript II RT have been optimized for yield and size of the cDNAs. To ensure that you obtain the best possible results, we suggest you read this section and the sections entitled Synthesizing the Second Strand and Ligating the attB1 Adapter before beginning.
cDNA synthesis is a multi-step procedure requiring many specially prepared reagents which are crucial to the success of the process. Quality reagents necessary for converting your mRNA sample into double-stranded cDNA are provided with this kit. To obtain the best results, do not substitute any of your own reagents for the reagents supplied with the kit.
Starting mRNA
To successfully construct a cDNA library, it is crucial to start with high-quality mRNA. For guidelines on isolating mRNA. The amount of mRNA needed to prepare a library depends on the efficiency of each step. Generally, 1 to 5 µg of mRNA will be sufficient to construct a cDNA library containing 106 to 107 primary clones in E. coli.
2.3 kb RNA Control
We recommend that you include the 2.3 kb RNA control in your experiments to help you evaluate your results. The 2.3 kb RNA control is an in vitro transcript containing the tetracycline resistance gene and its promoter (Tcr).
Guidelines
Consider the following points before performing the priming and first strand reactions:
Hot Start Reverse Transcription
Components of the first strand reaction are pre-incubated at 45°C before the addition of SuperScript II RT. Incubation at this temperature inhibits nonspecific binding of primer to template and reduces internal cDNA synthesis and extension by SuperScript II RT. For this reason, it is important to keep all reactions as close to 45°C as possible when adding SuperScript II RT.
If you are constructing multiple libraries, we recommend making a cocktail of reagents to add to each tube rather than adding reagents individually. This will reduce the time required for the step and will also reduce the chance of error.
Preparing [a-32 P]dCTP
If you will be labeling your first strand with [a-32 P]dCTP (10 µCi/µl), dilute an aliquot with DEPC-treated water to a final concentration of 1 µCi/µl. Use once and properly discard any unused portion as radioactive waste.
Using the Non-Radiolabeling Method
If you prefer to construct a non-radiolabeled cDNA library, perform the following protocols substituting DEPC-treated water for [a-32 P]dCTP.
Before Starting
You should have the following materials on hand before beginning. Keep all reagents on ice until needed.
Supplied with kit:
Supplied by user:
Diluting Your Starting mRNA
In a PCR tube or 1.5 ml tube, dilute your starting mRNA with DEPC-treated water according to the table below. The total volume for your mRNA + DEPC-treated water will vary depending on the amount of starting mRNA.
If you will be using the 2.3 kb RNA control supplied with the kit, add 5 µl of DEPC-treated water to 4 µl of the control mRNA for a total volume of 9 µl and a final mRNA amount of 2 µg.
µg of starting mRNA | ||||||
Reagent | ≤1 | 2 | 3 | 4 | 5 | Control |
mRNA + DEPC-treated water
|
10 µl
|
9 µl
|
8 µl
|
7 µl
|
6 µl
|
9 µl
(4 µl of mRNA + 5 µl of water)
|
Priming Reaction
µg of starting mRNA | ||||||
Reagent | ≤1 | 2 | 3 | 5 | Control | |
mRNA + DEPC-treated water
|
10 µl
|
9 µl
|
8 µl
|
7 µl
|
6 µl
|
9 µl
|
Biotin-
attB2-Oligo(dT) Primer (30 pmol/µl)
|
1 µl
|
1 µl
|
1 µl
|
1 µl
|
1 µl
|
1 µl
|
10 mM (each) dNTPs
|
1 µl
|
1 µl
|
1 µl
|
1 µl
|
1 µl
|
1 µl
|
Total Volume | 12 µl | 11 µl | 10 µl | 9 µl | 8 µl | 11 µl |
First Strand Reaction
µg of starting mRNA | ||||||
≤1 | 2 | 3 | 4 | 5 | Control | |
Total Volume
|
19 µl
|
18 µl
|
17 µl
|
16 µl
|
15 µl
|
18 µl
|
µg of starting mRNA | ||||||
≤1 | 2 | 3 | 4 | 5 | Control | |
SuperScript II RT (200 U/µl)
|
1 µl
|
2 µl
|
3 µl
|
4 µl
|
5 µl
|
2 µ
|
First Strand Reaction Sample
Follow the steps below to generate a sample for first strand analysis. We recommend analyzing the sample during an incubation step in the second strand reaction.
Synthesizing the Second Strand
Introduction
This section provides guidelines for synthesizing the second strand of cDNA. Perform all steps quickly to prevent the temperature from rising above 16°C.
Before Starting
You should have the following materials on hand before beginning. Keep all reagents on ice until needed.
Supplied with kit:
Supplied by user:
Size Fractionating Radiolabeled cDNA by Column Chromatography
Introduction
Column chromatography optimizes size fractionation of the cDNA and makes the cloning of larger inserts more probable. Follow instructions closely using the columns supplied with the kit to produce the highest quality library possible.
Use extreme caution when working with radioactive material. Follow all federal and state regulations regarding radiation safety.
How the Columns Work
Each column provided with the kit contains 1 ml of Sephacryl S-500 HR resin. This porous resin traps residual adapters and/or small cDNAs (<500 bp) and prevents them from contaminating the library. Larger molecules bypass the resin and elute quickly while smaller molecules are retained within the resin and elute more slowly. Thus, earlier eluted fractions contain larger cDNA fragments than later fractions.
If you are constructing more than one cDNA library, only add one cDNA adapter ligation reaction per column.
Before Starting
You should have the following materials on hand before beginning:
Supplied with kit:
Supplied by user:
Stopping the Ligation Reaction
1. Incubate the tube from step 2, at 70°C for 10 minutes to inactivate the ligase.
2. Place the tube on ice.
Setting Up the Column
Keep the following points in mind when setting up a fractionation column:
Washing the Column
cDNA size fractionation columns are packed in 20% ethanol which must be completely removed before adding your cDNA sample. Follow the steps below to remove the ethanol from the columns. The washing steps will take approximately 1 hour.
If the flow rate is noticeably slower than 30-40 seconds per drop, do not use the column. If the drop size from the column is not approximately 25 to 35 µl, do not use the column. The integrity and resolution of the cDNA may be compromised if the column does not meet these specifications.
Collecting Fractions
When collecting fractions, we recommend wearing gloves that have been rinsed with ethanol to reduce static.
Filling Out the Worksheet: Columns A, B, and C
A worksheet is provided to help you with your data recording (see the Appendix).
Using a pipet, measure the volume in each tube. Use a fresh tip for each fraction to avoid cross-contamination. Record this value in column A of the worksheet.
Filling Out the Worksheet: Columns D and E
Cerenkov counts will appear above background after approximately 300 µl of total volume (corresponding to fraction 5 in the sample worksheet).
Calculating the Double Strand cDNA Yield
Cerenkov counts are approximately 50% of the radioactivity that would be measured in scintillant. Use the specific activity (SA) determined from the first strand reaction sample and the equation below to calculate the yield of double-stranded cDNA.
Amount of ds cDNA (ng)
= (Cerenkov cpm) x 2 x (4 pmol dNTP/pmol dCTP) x (1000 ng/ µg ds cDNA)
SA (cpm/pmol dCTP) x (1515 pmol dNTP/µg ds cDNA)
= (Cerenkov cpm) x 8
SA x (1.515)
Required cDNA Yield
You will need a final cDNA yield of at least 30 ng to perform the BP recombination reaction. Because you will lose approximately half of your sample during the ethanol precipitation procedure, we recommend that you pool a minimum of 60 ng of cDNA from your fractions. See below for guidelines on selecting and pooling cDNA fractions.
Selecting and Pooling cDNA Fractions
The first fraction with detectable cDNA above background level contains the purest and largest cDNAs in the population. Because this fraction often does not contain enough cDNA for cloning, you may need to pool several fractions to reach a minimum of 60 ng of cDNA.
Note: The first 60 ng of cDNA from a column will make a library with a larger average insert size compared to a library made from the first 100 ng of cDNA. Use the values in column E to calculate the smallest volume needed from the next fraction to obtain the desired amount of cDNA for cloning.
Ethanol Precipitation
Calculating the cDNA Yield
Amount of ds cDNA (ng) = (Cerenkov cpm) x 8
SA x (1.515)
What You Should See
You should have a final cDNA yield of approximately 30-40 ng to perform the BP recombination reaction. Using approximately 30-40 ng of cDNA in the BP reaction should produce a library containing 5-10 million clones. If your cDNA yield is less than 30 ng, you may pool additional fractions and ethanol precipitate the cDNA. Resuspend any additional cDNA pellets using the cDNA sample from step 6, above. Once you have the desired amount of cDNA, proceed to Performing the BP Recombination Reaction with Radiolabeled cDNA.
Performing the BP Recombination Reaction with Radiolabeled cDNA
Introduction
General guidelines are provided below to perform a BP recombination reaction between your attB-flanked cDNA and pDONR 222 to generate a Gateway entry library. We recommend that you include a positive control and a negative control (no attB substrate) in your experiment to help you evaluate your results. For a map and a description of the features of pDONR 222, see the Appendix.
Resuspending pDONR™222
pDONR 222 is supplied as 6 µg of supercoiled plasmid, lyophilized in TE buffer, pH 8.0. To use, resuspend pDONR 222 plasmid DNA in 24 µl of sterile water to a final concentration of 250 ng/µl.
Propagating pDONR 222
If you wish to propagate and maintain pDONR 222, we recommend using Library Efficiency DB3.1 Competent Cells (Catalog no. 11782-018) from Invitrogen for transformation. The DB3.1 E. coli strain is resistant to CcdB effects and can support the propagation of plasmids containing the ccdB gene. To maintain the integrity of the vector, select for transformants in media containing 50 µg/ml kanamycin and 30 µg/ml chloramphenicol.
Note: DO NOT use general E. coli cloning strains including TOP10 or DH5a for propagation and maintenance as these strains are sensitive to CcdB effects. DO NOT use the ElectroMAX DH10B competent cells provided.
Positive Control
pEXP7-tet control DNA is included to use as a positive control for the BP reaction. pEXP7-tet contains an approximately 1.4 kb fragment consisting of the tetracycline resistance gene and its promoter (Tcr) flanked by attB sites. Using the pEXP7-tet fragment in a BP reaction with a donor vector results in entry clones that express the tetracycline resistance gene.
Recommended cDNA:pDONR™222 Ratio
For optimal results, we recommend using 30-40 ng of cDNA and 250 ng of pDONR 222 in a 10 µl BP recombination reaction. If the amount of cDNA you will be using is out of this range, make the following changes to the protocol below:
Before Starting
You should have the following materials on hand before beginning. Keep all reagents on ice until needed.
Supplied with kit:
Supplied by user:
BP Recombination Reaction
The following protocol uses 30-40 ng of cDNA and 250 ng of pDONR 222 in a 10 µl BP reaction. Use 30 ng of your 2.3 kb RNA control cDNA for the BP reaction. If the attB-flanked cDNA sample is greater than 4 µl, see below for necessary modifications.
Component | cDNA Sample | 2.3 kb RNA Control | BP Negative Control | BP Positive Control | |
attB-flanked cDNA (30-40 ng)
|
X µl
|
X µl
|
--
|
--
| |
pDONR 222 (250 ng/µl)
|
1 µl
|
1 µl
|
1 µl
|
1 µl
| |
pEXP7-tet positive control (50 ng/µl)
|
--
|
--
|
--
|
0.5 µl
| |
5X BP Clonase Reaction Buffer
|
2 µl
|
2 µl
|
2 µl
|
2 µl
| |
TE buffer, pH 8.0
|
to 7 µl
|
to 7 µl
|
4 µl
|
3.5 µl
|
Preparing for Transformation
Introduction
Once you have performed the BP recombination reaction, you will inactivate the reaction with proteinase K, ethanol precipitate the cDNA, and transform it into competent E. coli. The ElectroMAX DH10B T1 Phage Resistant Cells have a high transformation efficiency (>1 x 1010 cfu/µg DNA) making them ideal for generating cDNA libraries. Follow the guidelines below to prepare for the transformation procedure.
Transformation Control
pUC19 plasmid is included to check the transformation efficiency of ElectroMAX DH10B T1 Phage Resistant Cells. Transform 10 pg of pUC19 using the protocol.
Before Starting
You should have the following materials on hand before beginning:
Supplied with kit:
Supplied by user:
Stopping the BP Recombination Reaction
1. To each BP reaction from step 5, add 2 µl of proteinase K to inactivate the BP Clonase enzyme mix.
2. Incubate the reactions at 37°C for 15 minutes then at 75°C for 10 minutes.
Ethanol Precipitation
cDNA Library | 2.3 kb RNA Control | BP Negative Control | BP Positive Control | pUC 19 Control | |
Number of 1.5 ml Tubes |
6
|
2
|
2
|
2
|
1
|
Aliquot in Each Tube |
1.5 µl
|
1.5 µl
|
1.5 µl
|
1.5 µl
|
1.0 µl
|
cDNA Library | 2.3 kb RNA Control | BP Negative Control | BP Positive Control | pUC 19 Control | |
Dilutions
| |||||
Amount to Plate of Each Dilution
|
2 x 100 µl
|
2 x 100 µl
|
2 x 100 µl
|
2 x 100 µl
|
2 x 100 µl
|
Total Number of LB + Kan Plates
| 6 |
6
| 6 | 6 | -- |
Total Number of LB + Amp Plates
| -- | -- | -- | -- | 2 |
Determining the cDNA Library Titer
Introduction
Guidelines are provided below to determine the titer of your cDNA library.
Calculations
1. Using the results from the plating assay, and the equation below, calculate the titer for each plate.
cfu/ml = colonies on plate x dilution factor
volume plated (ml)
2. Use the titer for each plate to calculate the average titer for the entire cDNA library.
3. Use the average titer and the equation below to determine the total number of colony-forming units.
Total CFU (cfu) = average titer (cfu/ml) x total volume of cDNA library (ml)
Note: If you completed 6 electroporations for your cDNA library, the total volume will be 12 ml. For the controls, you will need to extrapolate the total number of colony-forming units using a total volume of 12 ml.
Expected Total CFUs
In general, a well represented library should contain 5 x 106 to 1 x 107 primary clones. If the number of primary clones is considerably lower for your cDNA library, see Troubleshooting.
What You Should See
See the table below for expected titers and expected total colony-forming units for the control reactions.
Expected Volume | |||
2.3 kb RNA control
|
≥ 1 x 10
6 cfu/ml
|
12 ml
|
≥ 1 x 10
7 cfu
|
BP positive control
|
≥ 1 x 10
6 cfu/ml
|
12 ml
|
≥ 1 x 10
7 cfu
|
BP negative control
|
≤ 0.3% of BP
positive control
|
12 ml
|
≤ 0.3% of BP positive control
|
pUC19 control
|
≥1 x 10
10 cfu/µg DNA
|
--
|
--
|
Qualifying the cDNA Library
Introduction
It is important to qualify the cDNA library to determine the success of your cDNA library construction. Determining the average insert size and percentage of recombinants will give you an idea of the representation of your cDNA library.
General Molecular Biology Techniques
For help with restriction enzyme analysis, DNA sequencing, and DNA biochemistry, refer to Molecular Cloning: A Laboratory Manual (Sambrook et al., 1989) or Current Protocols in Molecular Biology (Ausubel et al., 1994).
Before Starting
You should have the following materials on hand before beginning:
Supplied by user:
Analyzing Transformants by BsrG I Digestion
You will be digesting positive transformants with BsrG I to determine average insert size and percentage of recombinants. BsrG I sites generally occur at a low frequency making it an ideal restriction enzyme to use for insert size analysis. BsrG I cuts within the following sites:
Restriction Digest
We recommend that you analyze a minimum of 24 positive clones to accurately determine average insert size and the percentage of recombinants.
Expected Digestion Patterns
Use the following guidelines to determine the size of the cDNA inserts.
2.5 kb
1.4 kb
790 bp
Determining Average Insert Size and % Recombinants
What You Should See
You should see an average insert size of ≥1.5 kb and at least 95% recombinants for your cDNA library. If the average insert size or percent recombinants of your library clones is significantly lower, the cDNA going into the BP recombination reaction is either of poor quality or is insufficient in quantity. To troubleshoot any of the cDNA synthesis steps, see Troubleshooting.
The Next Step
If you wish to sequence entry clones, proceed to Sequencing Entry Clones.
You may screen your cDNA library to identify a specific entry clone and use this entry clone in an LR recombination reaction with a destination vector to generate an expression clone. Refer to the Gateway Technology manual to perform an LR recombination reaction using a single entry clone. Alternatively, you may transfer your cDNA library into a destination vector to generate an expression library for functional analysis. For detailed guidelines, refer to Performing the LR Library Transfer Reaction.
Sequencing Entry Clones
Introduction
You may sequence entry clones generated by BP recombination using any method of choice.
Sequencing Primers
To sequence inserts in entry clones derived from BP recombination with pDONR 222, we recommend using the following sequencing primers. Refer to the following table for the location of the primer binding sites.
Forward primer (proximal to attL1)
M13 Forward (-20): 5' -GTAAAACGACGGCCAG-3'
Reverse primer (proximal to attL2)
M13 Reverse: 5' -CAGGAAACAGCTATGAC-3'
The M13 Forward (-20) and M13 Reverse Primers (Catalog nos. N520-02 and N530-02, respectively) are available separately from Invitrogen. For other primers, Invitrogen offers a custom primer synthesis service. For more information, visit our Web site (www.thermofisher.com) or contact Technical Service.
Note: If you experience difficulty using the M13 Reverse Primer to sequence entry clones, we recommend using an alternative reverse primer that hybridizes to the poly A tail of your cDNA insert. Design your reverse primer such that it is 5' -(T 23N-3' where N is A, C, or G.
General Guidelines
The AT rich attL sites in the entry clones may decrease the efficiency of the sequencing reactions. To optimize your sequencing reactions, we recommend the following:
Recombination Region
The recombination region of the entry library resulting from pDONR 222 x attB-flanked cDNA is shown below.
Features of the Recombination Region:
Introduction
Column chromatography optimizes size fractionation of the cDNA and makes the cloning of larger inserts more probable. Follow instructions closely using the columns supplied with the kit to produce the highest quality library possible.
Because your cDNA is not labeled with [a-32 P]dCTP, you will need to estimate your cDNA yields using a plate spotting assay. You will be performing this assay throughout the size fractionation procedure. We recommend that you read the section entitled Performing the Plate Spotting Assay, before size fractionating your cDNA.
How the Columns Work
Each column provided with the kit contains 1 ml of Sephacryl S-500 HR resin. This porous resin traps residual adapters and/or small cDNAs (<500 bp) and prevents them from contaminating the library. Larger molecules bypass the resin and elute quickly while smaller molecules are retained within the resin and elute more slowly. Thus, earlier eluted fractions contain larger cDNA fragments than later fractions.
If you are constructing more than one cDNA library, only add one cDNA adapter ligation reaction per column.
Before Starting
You should have the following materials on hand before beginning:
Supplied with kit:
Supplied by user:
Stopping the Ligation Reaction
1. Incubate the tube from step 2, at 70°C for 10 minutes to inactivate the ligase.
2. Place the tube on ice.
Setting Up the Column
Keep the following points in mind when setting up a fractionation column:
Washing the Column
cDNA size fractionation columns are packed in 20% ethanol which must be completely removed before adding your cDNA sample. Follow the steps below to remove the ethanol from the columns. The washing steps will take approximately 1 hour.
If the flow rate is noticeably slower than 30-40 seconds per drop, do not use the column. If the drop size from the column is not approximately 25 to 35 µl, do not use the column. The integrity and resolution of the cDNA may be compromised if the column does not meet these specifications.
Collecting Fractions
When collecting fractions, we recommend wearing gloves that have been rinsed with ethanol to reduce static.
Filling Out the Worksheet: Columns A and B
A worksheet is provided to help you with your data recording.
Important: These fractions contain increasing amounts of the attB1 Adapter which will interfere with cloning reactions and will contaminate the library. We recommend discarding these tubes to avoid accidentally using them in the remainder of the protocol.
Filling Out the Worksheet: Columns C and D
You will be estimating the concentration and yield of your cDNA fractions using the plate spotting assay. Refer to Performing the Plate Spotting Assay for detailed guidelines on preparing the plates and staining the DNA.
Required cDNA Yield
You will need a final cDNA yield of 75 ng to perform the BP recombination reaction. Because you will lose approximately half of your sample during the ethanol precipitation procedure, we recommend that you pool a minimum of 150 ng of cDNA from your fractions. See below for guidelines on selecting and pooling cDNA fractions.
If you have previously performed the BP recombination reaction using radiolabeled cDNA, note that the amount of non-radiolabeled cDNA required is greater. This larger amount is due to the difference in scale between quantifying DNA by radioactivity using a scintillation counter and quantifying DNA by the plate spotting assay using the DNA standard. Thus, 30 ng of cDNA as measured by counts is roughly equivalent to 50-100 ng of cDNA as measured by comparison to the DNA standard.
Selecting cDNA Fractions
The first fractions containing detectable cDNA by the plate spotting assay contain the purest and largest pieces of cDNA in the population. You will want to use cDNA from these fractions for the BP recombination reaction.
We recommend that you also include the fraction preceding the first fraction with detectable cDNA. This fraction may contain large pieces of cDNA in quantities that are not visible using the plate spotting assay.
Pooling cDNA Fractions
You will need to pool fractions together to obtain approximately 150 ng of cDNA. Start with the fraction preceding the first fraction containing detectable cDNA. Add cDNA from subsequent fractions until the desired amount of cDNA is reached.
Note: The first 150 ng of cDNA from a column will make a library with a larger average insert size compared to a library made from the first 300 ng of cDNA. Use the values in column C to calculate the smallest volume needed from the next fraction to obtain the desired amount of cDNA for cloning.
Ethanol Precipitation
Preparing Aliquots for the Plate Spotting Assay
1. Remove 0.5 µl of your cDNA sample from step 6, above, and add to 4.5 µl of TE buffer to make a 1:10 dilution.
2. Remove 2.5 µl of the 1:10 dilution and add to 2.5 µl of TE buffer to make a 1:20 dilution.
Estimating the cDNA Yield
You will be estimating the concentration and yield of your cDNA sample using the plate spotting assay. Refer to Performing the Plate Spotting Assay, for detailed guidelines on preparing the plates and staining the DNA.
What You Should See
You should have a final cDNA yield of approximately 75-100 ng to perform the BP recombination reaction. Using approximately 75-100 ng of cDNA in the BP reaction should produce a library containing 5-10 million clones. If your cDNA yield is less than 75 ng, you may pool additional fractions and ethanol precipitate the cDNA. Resuspend any additional cDNA pellets using the cDNA sample from step 6, Ethanol Precipitation.
Once you have the desired amount of cDNA, proceed to Performing the BP Recombination Reaction with Non-Radiolabeled cDNA.
Introduction
General guidelines are provided below to perform a BP recombination reaction between your attB-flanked cDNA and pDONR 222 to generate a Gateway entry library. We recommend that you include a positive control and a negative control (no attB substrate) in your experiment to help you evaluate your results. For a map and a description of the features of pDONR 222.
Resuspending pDONR 222
pDONR 222 is supplied as 6 µg of supercoiled plasmid, lyophilized in TE buffer, pH 8.0. To use, resuspend pDONR 222 plasmid DNA in 24 µl of sterile water to a final concentration of 250 ng/µl.
Propagating pDONR™222
If you wish to propagate and maintain pDONR 222, we recommend using Library Efficiency DB3.1 Competent Cells (Catalog no. 11782-018) from Invitrogen for transformation. The DB3.1 E. coli strain is resistant to CcdB effects and can support the propagation of plasmids containing the ccdB gene. To maintain the integrity of the vector, select for transformants in media containing 50 µg/ml kanamycin and 30 µg/ml chloramphenicol.
Note: DO NOT use general E. coli cloning strains including TOP10 or DH5a for propagation and maintenance as these strains are sensitive to CcdB effects. DO NOT use the ElectroMAX DH10B competent cells provided with this kit.
Positive Control
pEXP7-tet control DNA is included with this kit for use as a positive control for the BP reaction. pEXP7-tet contains an approximately 1.4 kb fragment consisting of the tetracycline resistance gene and its promoter (Tcr) flanked by attB sites. Using the pEXP7-tet fragment in a BP reaction with a donor vector results in entry clones that express the tetracycline resistance gene.
Before Starting
You should have the following materials on hand before beginning. Keep all reagents on ice until needed.
Supplied with kit:
Supplied by user:
BP Recombination Reaction
The following protocol uses 75-100 ng of cDNA and 250 ng of pDONR™222 in a 10 µl BP reaction. If the attB-flanked cDNA sample is greater than 4 µl, see below for necessary modifications.
Component | cDNA Sample | 2.3 kb RNA Control | BP Negative Control | BP Positive Control | |
attB-flanked cDNA (75-100 ng)
|
X µl
|
X µl
|
--
|
--
| |
pDONR
™222 (250 ng/µl)
|
1 µl
|
1 µl
|
1 µl
|
1 µl
| |
pEXP7-tet positive control (50 ng/µl)
|
--
|
--
|
--
|
0.5 µl
| |
5X BP Clonase
™ Reaction Buffer
|
2 µl
|
2 µl
|
2 µl
|
2 µl
| |
TE buffer, pH 8.0
|
to 7 µl
|
to 7 µl
|
4 µl
|
3.5 µl
|
Performing a 20 µl BP Reaction
If you will be using more than 4 µl of cDNA, you may increase the total BP reaction volume to 20 µl. You will need to make the following changes to the above protocol:
Introduction
If you are constructing a non-radioactive cDNA library, you will be estimating your cDNA yields using a plate spotting assay. Samples will be spotted on agarose and compared under UV light to spots containing known quantities of DNA. Guidelines are provided below to prepare the plates and to perform the assay.
The plate spotting assay is an assay to qualitatively determine the concentration and yield of your cDNA samples. Comparison of samples to the DNA standard is subjective and may vary from person to person. In addition, the plate spotting assay is limited in its range of cDNA detection. While you can detect as little as 1 ng of cDNA using SYBR Gold Nucleic Acid Gel Stain (see Choosing a Nucleic Acid Stain, below), the assay cannot resolve an unlimited amount of cDNA. Generally, spots containing more than 50 ng of cDNA will appear equally stained under UV light.
Choosing a Nucleic Acid Stain
DNA may be detected using ethidium bromide or SYBR Gold Nucleic Acid Gel Stain available from Molecular Probes (Catalog no. S11494). We recommend using SYBR Gold because it is 10-fold more sensitive than ethidium bromide for detecting DNA in electrophoretic gels.
Ethidium bromide staining requires preparing plates containing agarose plus ethidium bromide. SYBR Gold staining requires preparing agarose-only plates followed by staining the plate using a SYBR Gold solution. Guidelines are provided in this section for both stains.
Using the pEXP7-tet Positive Control
Supercoiled pEXP7-tet DNA is included with the kit as a positive control for the BP recombination reaction. pEXP7-tet can also be used as a DNA standard for the plate spotting assay. The concentration of your cDNA samples can be estimated by comparison under UV light to known quantities of pEXP7-tet DNA.
Number of Plates Needed
You will need two plates per library. One plate will contain each of your fractions and another plate will contain cDNA samples that were pooled and ethanol precipitated.
Before Starting
You should have the following materials on hand before beginning.
Supplied with kit:
Supplied by user:
Preparing Plates
Preparing pEXP7-tet Control DNA
Serially dilute pEXP7-tet control DNA in TE buffer to final concentrations of:
25 ng/µl
10 ng/µl
5 ng/µl
1 ng/µl
DNA standards can be stored at -20°C for up to 1 month.
Labeling Plates
Using a marker, label plates on the bottom side of the petri dish and indicate where the DNA standards and samples will be spotted (see below).
Sample Plates for cDNA Size Fractionation by Column Chromatography
Guidelines
Consider the following points before performing the DNA plate spotting assay:
DNA Spotting Assay
Staining Plates with SYBR Gold
Introduction
Once you have qualified your cDNA library and analyzed entry clones, you can perform the LR recombination reaction to transfer your cDNA library into any Gateway destination vector of choice. If you will be creating an expression library, you will need to follow the guidelines provided in this section for preparing DNA and for performing the LR recombination reaction.
Alternatively, you may screen your cDNA library to identify a specific entry clone and use this entry clone in an LR recombination reaction with a destination vector to generate an expression clone. Refer to the Gateway Technology manual to perform a standard LR recombination reaction using a single entry clone.
Before Starting
You should have the following materials on hand before beginning.
Supplied with kit:
Supplied by user:
Preparing Double-Stranded DNA
You may prepare plasmid DNA from your cDNA library using your method of choice. We recommend using the S.N.A.P. MidiPrep Kit (Catalog no. K1910-01). Consider the following points when preparing your DNA:
PEG Precipitation
After you have prepared plasmid DNA from your cDNA library, precipitate the DNA using the 30% PEG/Mg solution provided with the kit.
Determining the DNA Yield
Component | Sample | Negative Control | Positive Control | |
cDNA entry library (25 ng/µl)
|
2 µl
|
--
|
--
| |
Positive control plasmid (25 ng/µl)
|
--
|
--
|
2 µl
| |
Destination vector (150 ng/µl)
|
3 µl
|
3 µl
|
3 µl
| |
5X LR Clonase
™ Reaction Buffer
|
4 µl
|
4 µl
|
4µl
| |
TE Buffer, pH 8.0
|
5 µl
|
7 µl
|
5 µl
| |
Total volume | 14 µl | 14 µl | 14 µl |
The following table lists some potential problems and possible solutions that may help you troubleshoot various steps during cDNA library construction.
Note that the starting mRNA quality is a key factor that will affect the outcome of your results.
Problem | Cause | Solution |
---|---|---|
Low cDNA yield or low incorporation of [a-32P]dCTP after first strand synthesis (radiolabeling method only) | Insufficient starting mRNA | Quantitate the mRNA by measuring the A260, if possible. We recommend using 1-5 mg of starting mRNA. |
Poorly prepared mRNA or degraded mRNA | Follow the recommendations for mRNA isolation and working with mRNA. | |
Old [a-32P]dCTP or [a-32P]dCTP not added | Do not use [a-32P]dCTP that is more than 2 weeks old. Use fresh [a-32P]dCTP. See guidelines on preparing [a-32P]dCTP. | |
Essential reagent accidentally not added or not working | Perform the 2.3 kb RNA control reaction to verify that the correct reagents have been added and are working properly. | |
Inaccurate incubation temperatures or temperature fluctuations | Perform the first strand reaction at 45°C. Keep reactions at 45°C when adding SuperScript II RT. | |
SuperScript II RT stored incorrectly | Store SuperScript II RT at -20°C in a frost-free freezer. | |
Low cDNA yield after size fractionation by column chromatography | Faulty columns | Check each column to verify that it is working properly. |
Samples run too quickly over columns | Let columns drain completely before adding additional buffer. | |
Low cDNA library titer with pUC19 transformation control working properly | cDNA of poor quality | Make sure the first strand reaction shows >15% percent incorporation of [a-32P]dCTP (radiolabeling method only). |
Insufficient ligation of attB1 Adapter | Perform the 2.3 kb RNA control reactions to verify the ligation step worked properly. | |
Incorrect ratio of cDNA to pDONR 222 | Refer to the recommended ratio of cDNA to pDONR 222 for the BP reaction. | |
Low cDNA library titer with pUC19 transformation control working properly, continued | Insufficient amount of cDNA used in the BP recombination reaction | Use the minimum amount of cDNA required for the BP recombination reaction. Refer to the radiolabeling method and the non-radiolabeling method. |
BP Clonase enzyme mix is inactive or suggested amount was not used |
| |
Few or no colonies obtained from the pUC19 transformation control | Recombination reactions were not treated with proteinase K | Treat reactions with proteinase K before transformation. |
Introduction
In this section, we provide a sample experiment to illustrate the cDNA library construction process. This experiment starts with isolated mRNA and continues through construction and qualification of a radiolabeled cDNA library. All steps were performed according to these protocols.
Starting mRNA
3 µg of high-quality HeLa cell mRNA
First Strand Analysis
A sample of the first strand reaction was removed and analyzed to determine specific activity, cDNA yield, and percent incorporation of [a-32P]dCTP. The unwashed and washed filters gave the following counts:
Specific Activity
The specific activity was determined using the counts for the unwashed filter and the equation below:
Counts per Minute (cpm)
| |
Unwashed Filter
|
45998
|
Washed Filter
|
2601
|
SA (cpm/pmol dCTP) = (cpm unwashed filter/10 µl
200 pmol dCTP/10 µl
= 45998 cpm/10 µl
200 pmol dCTP/10 µl
= 230 cpm/pmol dCTP
First Strand cDNA Yield
The first strand cDNA yield was determined using the counts for the washed filter, the calculated specific activity, and the equation below:
cDNA Yield (µg)
= (cpm of washed filter) x (25 µl/10 µl) x (20 µl/1 µl) x (4 pmol dNTP/pmol dCTP)
SA (cpm/pmol dCTP) (3030 pmol dNTP/ µg cDNA)
= (cpm of washed filter) x 50 x (4 pmol dNTP/pmol dCTP)
SA (cpm/pmol dCTP) (3030 pmol dNTP/ µg cDNA)
= (cpm of washed filter) x (200)
SA x (3030)
= 2601 x 200
230 x 3030
= 0.746 µg cDNA
Percent Incorporation
The percent incorporation of [a-32P]dCTP was determined using the calculated first strand cDNA yield and the equation below:
Percent Incorporation = cDNA yield (µg) x 100
starting mRNA amount (µg)
= 0.746 µg cDNA x 100
3 µg starting mRNA
= 25%
The results of the first strand analysis are summarized below:
Specific Activity 230 cpm/pmol dCTP
cDNA Yield 0.746 µg
Percent Incorporation 25%
Size Fractionation by Column Chromatography
After attB1 adapter ligation, the cDNA was size fractionated using column chromatography. The results are listed in the sample worksheet below. Tube 5 was the first tube to give Cerenkov counts above background. Using the data for tube 5, we demonstrate below how the worksheet was filled out.
Tube 5 Example
The volume in tube 5 was measured to be 36 µl (column A). Adding this volume to the previous cumulative volume (i.e. 306 µl) gave a total volume of 342 µl (column B). The Cerenkov count was 213 cpm (column C).
The double strand cDNA yield was determined using the count value from column C, the specific activity already calculated in the first strand analysis, and the equation below:
Amount of ds cDNA (ng)
= (Cerenkov cpm) x 2 x (4 pmol dNTP/pmol dCTP) x (1,000 ng/ µg ds cDNA)
SA (cpm/pmol dCTP) x (1515 pmol dNTP/ µg ds cDNA)
= (Cerenkov cpm) x 8
SA x (1.515)
= 213 x 8
230 x 1.515
= 4.9 ng cDNA (column D)
The concentration of cDNA was determined using the calculated cDNA yield and the value in column A.
Concentration of cDNA (ng/µl) = amount of cDNA (ng)
fraction volume (µl)
= Column D
Column A
= 4.9 ng
36 µl
= 0.136 ng/µl (column E)
Sample cDNA Library Worksheet
Tube | A Fraction Volume (µl) | B Total Volume (µl) | C Cerenkov Counts (cpm) | D Amount of cDNA (ng) | E Concentration of cDNA (ng/µl) |
1 | 151 | 151 | 22 | -- | -- |
2 | 85 | 236 | 14 | -- | -- |
3 | 34 | 270 | 25 | -- | -- |
4 | 36 | 306 | 15 | -- | -- |
5 | 36 | 342 | 213 | 4.9 | 0.136 |
6 | 34 | 376 | 1136 | 26.1 | 0.77 |
7 | 35 | 411 | 2628 | 60.3 | 1.72 |
8 | 36 | 447 | 4114 | 94.5 | 2.625 |
9 | 36 | 483 | 4427 | 101.6 | 2.82 |
10 | 33 | 516 | 3614 | 83.0 | 2.52 |
11 | 36 | 552 | 2947 | 67.7 | 1.88 |
12 | 36 | 588 | 2139 | 49.1 | 1.36 |
13 | 36 | 624 | 1761 | 40.4 | 1.12 |
14 | 36 | ||||
15 | 36 | ||||
16 | 35 | ||||
17 | 36 | ||||
18 | 36 | ||||
19 | 36 | ||||
20 | 36 |
Selecting and Pooling Fractions
Fractions 5, 6, and part of fraction 7 were pooled together for a total of 61.1 ng of cDNA (see table below).
Fraction | Pooled Volume (µl) | Concentration of cDNA (ng/µl) | Amount of cDNA (ng) |
5 | 36 | 0.136 | 4.9 |
6 | 34 | 0.77 | 26.1 |
7 | 17.5 | 1.72 | 30.1 |
Total Pooled cDNA (ng) | 61.1 |
Calculating the cDNA Yield
After ethanol precipitation, the pooled cDNA gave a Cerenkov count of 1538 cpm. cDNA yield was determined using the count value, th
Size Fractionation by Column Chromatography
A sample plate and worksheet is provided below to demonstrate how to estimate the yield of your non-radiolabeled cDNA. Samples were size fractionated by column chromatography and cDNA yields were estimated using the plate spotting assay. Refer to Labeling Plates, in Performing the Plate Spotting Assay to see how plates were labeled. Note that samples are in the reverse order.
Serial dilutions of pEXP7-tet control DNA and column fractions 1-13 were spotted and stained with SYBR Gold as described.
Tube | A Fraction Volume (µl) | B Total Volume (µl) | C Concentration of cDNA (ng/µl) | D Amount of cDNA (ng) |
1 | 151 | 151 | -- | -- |
2 | 85 | 236 | -- | -- |
3 | 34 | 270 | -- | -- |
4 | 36 | 306 | -- | -- |
5 | 36 | 342 | 0.5 | 18 |
6 | 34 | 376 | 4 | 136 |
7 | 35 | 411 | 8 | 280 |
8 | 36 | 447 | 10 | 360 |
9 | 36 | 483 | -- | -- |
10 | 33 | 516 | -- | -- |
11 | 36 | 552 | -- | -- |
12 | 36 | 588 | -- | -- |
13 | 36 | 624 | -- | -- |
14 | 36 | |||
15 | 36 | |||
16 | 35 | |||
17 | 36 | |||
18 | 36 | |||
19 | 36 | |||
20 | 36 |
Selecting and Pooling Fractions
Fractions 5, 6, and part of fraction 7 were pooled together for a total of 294 ng of cDNA (see table below).
Fraction | Pooled Volume (µl) | Concentration of cDNA (ng/µl) | Amount of cDNA (ng) |
---|---|---|---|
5 | 36 | 0.5 | 18 |
6 | 34 | 4 | 136 |
7 | 17.5 | 8 | 140 |
Total Pooled cDNA (ng) | 294 |
Estimating the cDNA Yield
After ethanol precipitating the pooled cDNA, cDNA yield was estimated using the plate spotting assay. Refer to Labeling Plates, in Performing the Plate Spotting Assay to see how plates were labeled. Note that samples are in the reverse order.
Serial dilutions of pEXP7-tet control DNA and two dilutions of ethanol-precipitated cDNA were spotted and stained with SYBR Gold as described.
1:10 Dilution | 1:20 Dilution | |
---|---|---|
cDNA Concentration of Diluted Sample (ng/µl) | 5 | 2.5 |
Final cDNA concentration (ng/µl) | 50 | 50 |
Volume of cDNA (µl) | 4 | 4 |
Total cDNA Yield (ng) | 200 | 200 |
BP Recombination Reaction
3 µl of the cDNA sample containing a total of 150 ng of cDNA was used in the BP recombination reaction.
Introduction
A worksheet is provided to help you with your record keeping and calculations. Before you record any data, we suggest you make several copies of this worksheet for use with additional cDNA synthesis reactions.
Tube | A Fraction Volume (µl) | B Total Volume (µl) | C Cerenkov Counts (cpm) | D Amount of cDNA (ng) | E Concentration of cDNA (ng/µl) |
1 | |||||
2 | |||||
3 | |||||
4 | |||||
5 | |||||
6 | |||||
7 | |||||
8 | |||||
9 | |||||
10 | |||||
11 | |||||
12 | |||||
13 | |||||
14 | |||||
15 | |||||
16 | |||||
17 | |||||
18 | |||||
19 | |||||
20 |
Introduction
A worksheet is provided to help you with your record keeping and calculations. Before you record any data, we suggest you make several copies of this worksheet for use with additional cDNA synthesis reactions.
Tube | A Fraction Volume (µl) | B Total Volume (µl) | C Concentration of cDNA (ng/µl) | D Amount of cDNA (ng) |
1 | ||||
2 | ||||
3 | ||||
4 | ||||
5 | ||||
6 | ||||
7 | ||||
8 | ||||
9 | ||||
10 | ||||
11 | ||||
12 | ||||
13 | ||||
14 | ||||
15 | ||||
16 | ||||
17 | ||||
18 | ||||
19 | ||||
20 |
Features of the Vector
pDONR 222 (4718 bp) contains the following elements. All features have been functionally tested.
Feature | Benefit |
rrnB T1 and T2 transcription terminators
|
Protects the cloned gene from expression by vector-encoded promoters, thereby reducing possible toxicity (Orosz
et al., 1991).
|
M13 forward (-20) priming site
|
Allows sequencing in the sense orientation.
|
attP1 and
attP2 sites
|
Bacteriophage λ-derived DNA recombination sequences that permit recombinational cloning of
attB-containing cDNA (Landy, 1989).
|
BsrG I restriction sites
|
Allows detection and size determination of cDNA inserts by restriction enzyme analysis.
|
ccdB gene
|
Allows negative selection of the plasmid.
|
Chloramphenicol resistance gene
|
Allows counterselection of the plasmid.
|
M13 reverse priming site
|
Allows sequencing in the anti-sense orientation.
|
Kanamycin resistance gene
|
Allows selection of the plasmid in
E. coli.
|
pUC origin
|
Allows high-copy replication and maintenance of the plasmid in
E. coli.
|
For Research Use Only. Not for use in diagnostic procedures.