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The TA Cloning® Kit with pCR®2.1 provides a quick, one-step cloning strategy for the direct insertion of a PCR product into a plasmid vector.
Advantages
Using the TA Cloning® Kit:
How TA Cloning® Works
Taq polymerase has a non template-dependent activity that adds a single deoxyadenosine (A) to the 3´ ends of PCR products. The linearized vector supplied in this kit has single 3´ deoxythymidine (T) residues. This allows PCR inserts to ligate efficiently with the vector.
Diagram
The diagram below shows the concept behind the TA Cloning® method.
Thermostable polymerases containing extensive 3´ to 5´ exonuclease activity, such as Platinum® Pfx, do not leave 3´ A-overhangs. PCR products generated with Taq polymerase have a high efficiency of cloning in the TA Cloning® system as the 3´ A-overhangs are not removed. However, if you use a proofreading polymerase or wish to clone blunt-ended fragments, you can add 3´ A-overhangs by incubation with Taq at the end of your cycling program.
Alternatively, you may want to try the Zero Blunt® PCR Cloning Kit (Catalog nos. K2700-20 and K2750-20). This kit offers efficient cloning of blunt-end PCR products generated using thermostable, proofreading polymerases. For more information, visit our website or contact Technical Service.
Storage Instructions
The TA Cloning® Kits are shipped on dry ice and contain a box of TA Cloning® Reagents (Box 1) and a box of One Shot® Competent Cells (Box 2). Catalog nos. K2020-20 and K2020-40 are not supplied with One Shot® Competent Cells. Store Box 1 at -20°C in a non-frost-free freezer and Box 2 at -80°C.
TA Cloning® Reagents
TA Cloning® Reagents (Box 1) are listed below. Note that the user must supply Taq Polymerase. Forty reaction kits are supplied as two 20 reaction kits. Store Box 1 at -20ºC.
Item | Composition | Amount |
pCR
®2.1, linearized
|
25 ng/µl in 10 mM Tris-HCl, 1 mM EDTA, pH 8
|
5 x 10 µl
|
10X PCR Buffer
|
100 mM Tris-HCl, pH 8.3 (at 42°C)
500 mM KCl
25 mM MgC12
0.01% gelatin
|
100 µl
|
10X Ligation Buffer
|
60 mM Tris-HCl, pH 7.5
60 mM MgCl2
50 mM NaCl
1 mg/ml bovine serum albumin
70 mM β-mercaptoethanol
1 mM ATP
20 mM dithiothreitol
10 mM spermidine
|
100 µl
|
50 mM dNTPs
|
12.5 mM dATP
12.5 mM dCTP
12.5 mM dGTP
12.5 mM dTTP
(adjusted to pH 8.0)
|
10 µl
|
T4 DNA Ligase
|
4.0 Weiss units/µl
|
25 µl
|
Water
|
Deionized, autoclaved water
|
1 ml
|
Control DNA Template
|
0.1 µg/µl in 10 mM Tris-HCl, 1 mM EDTA, pH 8
|
10 µl
|
Control PCR Primers
|
0.1 µg/µl each in 10 mM Tris-HCl, 1 mM EDTA, pH 8
|
10 µl
|
One Shot® Reagents
The table below describes the items included in the One Shot® competent cell kit. Catalog nos. K2020-20 and K2020-40 are not supplied with competent cells. Forty reaction kits are supplied as two 20 reaction kits.
The transformation efficiency for TOP10F´ and TOP10 cells is 1 x 109 cfu/µg DNA. The transformation efficiency for INVaF´ is 1 x 108 cfu/µg DNA. Store competent cells at -80ºC.
Component | Composition | Amount |
S.O.C. Medium
(may be stored at room temperature or +4ºC)
|
2% Tryptone
0.5% Yeast Extract
10 mM NaCl
2.5 mM KCl
10 mM MgCl2
10 mM MgSO4
20 mM glucose (dextrose)
|
6 ml
|
INVaF´, TOP10F´,
or TOP10 cells
|
--
|
21 x 50 µl
|
pUC19 Control DNA
|
10 pg/µl in 5 mM Tris-HCl, 0.5 mM EDTA, pH 8
|
50 µl
|
Genotype of INVaF´
F´ endA1 recA1 hsdR17 (rk- , mk+ ) supE44 thi-1 gyrA96 relA1 f80lacZD M15 D (lacZYA-argF)U169 l-
Genotype of TOP10F´
F´ [lacIq Tn10 (TetR)] mcrA D (mrr-hsdRMS-mcrBC) F80lacZD M15 D lacC74 recA1 araD139 D(ara-leu)7697 galU galK rpsL (StrR ) endA1 nupG
Genotype of TOP10
F- mcrA D (mrr-hsdRMS-mcrBC) F80lacZDM15 D lacC74 recA1 araD139 D (ara-leu)7697 galU galK rpsL (StrR ) endA1 nupG
Introduction
To clone your gene of interest into pCR® 2.1, you must first generate a PCR product. The PCR product is ligated into pCR® 2.1 and transformed into competent cells. Since the PCR product can ligate into the vector in either orientation, individual recombinant plasmids need to be analyzed to confirm proper orientation. The correct recombinant plasmid is then purified for further subcloning or characterization.
Flow Chart
The table below describes the major steps necessary to clone your gene of interest into pCR® 2.1.
Step | Action | ||
1
|
Amplify your PCR product using
Taq polymerase and your own primers and parameters.
| ||
2
|
Ligate your PCR product into pCR
® 2.1.
| ||
3
|
Transform your ligation into competent
E. coli.
| ||
4
|
Select colonies and isolate plasmid DNA. Analyze plasmid DNA for the presence and orientation of the PCR product by restriction enzyme digestion or sequencing.
|
When using the TA Cloning® Kit for the first time, we recommend that you perform the control reactions to help you evaluate your results.
Guidelines for PCR
Generally 10-100 ng of DNA is sufficient to use as a template for PCR. If amplifying a pool of cDNA, the amount needed will depend on the relative abundance of the message of interest in your mRNA population. For optimal ligation efficiencies, we recommend using no more than 30 cycles of amplification.
Materials Supplied by the User
You will need the following reagents and equipment.
Polymerase Mixtures
If you wish to use a mixture containing Taq polymerase and a proofreading polymerase, Taq must be in excess of a 10:1 ratio to ensure the presence of 3’ A-overhangs on the PCR product. We recommend using our Platinum® Taq DNA Polymerase High Fidelity.
If you use polymerase mixtures that do not have enough Taq polymerase or a proofreading polymerase only, you can add 3’ A-overhangs using the method in the Appendix.
Producing PCR Products
Perform the PCR in a 50 µl volume containing:
DNA Template | 0-100 ng |
10X PCR Buffer | 5 µl |
50 mM dNTPs | 0.5 µl |
Primers | 1 µM each |
Water | to a total volume of 49 µl |
Taq Polymerase | 1 unit |
Total Volume | 50 µl |
Gel Purification
If you do not obtain a single, discrete band from your PCR, you may gel-purify your fragment before proceeding. Take special care to avoid sources of nuclease contamination and long exposure to UV light. Alternatively, you may optimize your PCR to eliminate multiple bands and smearing (Innis et al., 1990). Our PCR Optimizer™ Kit (Catalog no. K1220-01) can help you optimize your PCR. Contact Technical Service for more information.
Analyzing Positive Clones
Sequencing Your Insert
If you wish to sequence your insert in pCR® 2.1, you may use the M13 Reverse Primer to sequence into your insert from the lac promoter. To sequence into the insert from the lacZa fragment, you can use either the T7 Promoter Primer or the M13 Forward Primer. Refer to the map of pCR® 2.1 for the primer sequences and location of the primer binding sites. For your convenience, we offer a custom primer synthesis service.
If you have problems obtaining transformants or the correct insert, perform the control reactions as described. These reaction swill help you troubleshoot your experiment. Refer to the Troubleshooting section for additional tips.
Long-Term Storage
Once you have identified the correct clone, be sure purify the colony and make a glycerol stock for long-term storage. We recommend that you store a stock of plasmid DNA at -20°C.
1. Streak the original colony on LB plates containing 100 µg/ml ampicillin or 50 µg/ml kanamycin.
2. Isolate a single colony and inoculate into 1-2 ml of LB containing 100 µg/ml ampicillin or 50 µg/ml kanamycin.
3. Grow until culture reaches stationary phase.
4. Mix 0.85 ml of culture with 0.15 ml of sterile glycerol and transfer to a cryovial.
5. Store at -80°C.
If you do not obtain the results you expect, use the following table to troubleshoot your experiment. We recommend performing the control reactions to help you evaluate your results.
Problem | Reason | Solution |
No colonies obtained from transformation
|
Bacteria were not competent.
|
Use the pUC19 control vector included with the One Shot
® kit to test transformation efficiency.
|
Incorrect concentration of antibiotic on plates or the plates are too old.
|
Use 100 µg/ml of ampicillin or 50 µg/ml kanamycin. Use fresh ampicillin plates (< 1 month old).
| |
White colonies do not have insert
|
Single 3´ T-overhangs on the vector degraded.
|
Use another tube of vector. Avoid storing the vector for longer than 6 months or subjecting it to repeated freeze/thaw cycles. Check vector by performing the Self-Ligation Reaction.
|
Only white colonies obtained
|
No IPTG or X-Gal in plates.
|
Be sure to include X-Gal for blue/white screening and both IPTG and X-Gal if using TOP10F´.
|
Majority of colonies are blue or light blue with very few white colonies
|
The insert does not interrupt the reading frame of the
lacZ gene.
|
If you have a small insert (< 500 bp), you may have light blue colonies. Analyze blue colonies as they may contain insert.
|
Used a polymerase that does not add 3´ A-overhangs.
|
Do not use proofreading polymerases such as Platinum
®Pfx as they do not add 3´ A-overhangs. Use
Taq polymerase.
| |
PCR products were gel-purified before ligation.
|
Gel purification can remove the single 3´ A-overhangs. If gel purification is needed, use nuclease-free solutions to purify fragment or optimize your PCR.
| |
The PCR products were stored for a long period of time before performing the ligation reaction.
|
Use fresh PCR products. Efficiencies are reduced after as little as 1 day of storage.
| |
Too much of the amplification reaction was added to the ligation.
|
The high salt content of PCR reactions can inhibit ligation. Do not use more than 2-3 µl of the PCR reaction in the ligation reaction.
| |
Incorrect molar ratio of vector:insert used in the ligation reaction.
|
Estimate the concentration of the PCR product. Set up the ligation reaction with a 1:1 or 1:3 vector:insert molar ratio.
| |
Some colonies have a light blue color or appear white with blue centers
|
Leaky expression of the
lacZ fragment or only a partial disruption of
lacZ by the insert.
|
If you are looking for a smaller size insert, 500 bp or less, analyze these colonies as they may contain insert.
|
White colonies or blue colonies of normal size are surrounded by smaller, white colonies
|
The smaller colonies are ampicillin-sensitive satellite colonies. Do not pick the small colonies as they do not contain any plasmid.
|
Use kanamycin selection. Be sure the stock solution of ampicillin and your plates are both fresh.
|
White colonies do not grow in liquid culture
|
Ampicillin-sensitive satellite colonies.
|
Be sure to pick large white colonies. Be sure the ampicillin is fresh. Use kanamycin to eliminate this problem.
|
No results from sequencing
|
Accidental use of the amplification primers in the kit for sequencing. These are for generating the control PCR product only.
|
Use the M13 Forward (-20) and Reverse Primers for sequencing. You may also use the T7 promoter primer to sequence into the insert.
|
The T7 primer used was not the right sequence.
|
Check the sequence of your T7 promoter primer and make sure it matches with the priming site on pCR
® 2.1.
| |
An Sp6 primer was used to sequence inserts in pCR
® 2.1.
|
Do not use an Sp6 primer to sequence pCR
® 2.1. There is no binding site for this primer.
| |
No PCR product
|
Either the
Taq polymerase is inactive or the conditions for your PCR are not optimal.
|
Perform the control reactions to test the activity of the
Taq polymerase. If
Taq polymerase is active, you may need to optimize the conditions for your PCR reaction.
|
Low plasmid yield
|
Cells do not grow well in LB.
|
Try using S.O.C. medium with the appropriate antibiotic.
|
Explanation of Control Reactions
The following table describes the control reactions that can be performed to troubleshoot your TA Cloning® experiment and how to interpret the results from these control reactions.
Control Reaction | Explanation |
Self- Ligation
|
This control reaction shows if pCR
® 2.1 has lost the 3´ T overhangs. Loss of the T overhangs results in blunt end ligation and disruption of the
lacZa reading frame. False white colonies will result. Normally, less than 5% of the colonies should be white.
|
Transformation Control
|
Tests the transformation efficiency of the One Shot
® Competent Cells.
Transformation efficiency should be 1 x 10
8 cfu/µg DNA for INVF'and 1 x 10
9 cfu/µg DNA for TOP10 and TOP10F'.
|
Control PCR Product
|
Tests the PCR reagents including
Taq polymerase.
|
Control Ligation Reaction
|
Tests the ligation reagents and pCR
® 2.1. Greater than 80% white colonies are produced and these colonies should contain vector with insert.
|
Performing the Self-Ligation Reaction
The TA Cloning® vector is stable for six months if not subjected to repeated freeze-thaw cycles. Vector that has been stored for longer periods or repeatedly frozen and thawed will lose the 3´ T-overhangs resulting in "false" white positives. Follow the protocol below to perform the self-ligation reaction and transform One Shot® Competent Cells. If you are using another E. coli strain, follow the manufacturer’s instructions.
Procedure
Step | Time | Temperature | Cycles |
Denaturation
|
1 minute
|
94°C
|
25X
|
Annealing
|
1 minute
|
55°C
| 25X |
Extension
|
1 minute
|
72°C
| 25X |
Final Extension
|
7 minutes
|
72°C
|
1X
|
Feature | Benefit |
---|---|
lac promoter | Allows bacterial expression of the lacZa fragment for a-complementation (blue-white screening). |
lacZa fragment | Encodes the first 146 amino acids of ß-galactosidase. Complementation in trans with the W fragment gives active ß-galactosidase for blue-white screening. |
Kanamycin resistance gene | Allows selection and maintenance in E. coli, useful when cloning products amplified from ampicillin-resistant plasmids. |
Ampicillin resistance gene | Allows selection and maintenance in E. coli. |
pUC origin | Allows replication, maintenance, and high copy number in E. coli. |
T7 promoter and priming site | Allows in vivo or in vitro transcription of anti-sense RNA. Allows sequencing of the insert. |
M13 Forward (-20) and M13 Reverse Priming Sites | Allows sequencing of the insert. |
f1 origin | Allows rescue of sense strand for mutagenesis and single-strand sequencing. |
仅供科研使用,不可用于诊断目的。