Protocols

Introduction

We offer wide variety of pre-cast gels. These include gels for analysis nucleic acids (TBE, TBE-Urea, and DNA Retardation).  General information on Novex® Pre-Cast Gels is provided in this section.   Novex® Pre-Cast Gels are capable of resolving proteins in the range of 2-500 kDa and nucleic acids in the range of 10-3000 bp.
 
Choosing a Gel for Your Application

To obtain the best results for your application, it is important to choose the correct gel percentage, buffer system, gel format, and thickness. Many factors affect the choice of a gel. These include:
 
Application

Based on the type of your application, you can choose from gels for protein separation (Tris-Glycine, Tricine, IEF, ZOOM® and Zymogram Gels) or gels for nucleic acid separation (TBE, TBE-Urea, and DNA Retardation Gels).
 
Size of the molecule being separated

Large molecules resolve well on a low percentage gels while small molecules are best resolved on high percentage gels. The size of the molecule usually dictates the acrylamide percentage. If you do not know the molecular weight of the molecule or are separating a wide molecular weight range of molecules, choose gradient gels.
 
Amount of available material

The higher the number of wells and the thinner the gel, the lower the sample loading volume and vice versa. Based on the amount of your starting material available, you can choose from a variety of comb types.

Compatibility

The size of a Novex® Pre-Cast Gel is 10 x 10 cm (gel size is 8 x 8 cm).  We recommend using the XCell SureLock™ Mini-Cell for the electrophoresis of Novex® Pre-Cast Gels to obtain optimal and consistent performance.  Novex® Pre-Cast Gels are compatible with most other mini-cells designed for electrophoresis of 10 cm (h) x 10 cm (w) gel cassettes. 

The Novex® Pre-Cast Gels are compatible with most silver staining protocols.  We recommend using the SilverQuest™ Silver Staining Kit or the SilverXpress® Silver Staining Kit for silver staining of Novex® Gels.  The Novex® Pre-Cast Gels are compatible with any of the standard Coomassie® staining procedures.  The protocols that are accelerated by heat are preferable as heat serves as a “fix” for proteins, especially smaller peptides.  The SimplyBlue™ SafeStain and Novex® Colloidal Coomassie® Blue Staining Kit  are recommended for staining Novex® Gels.

Applications
 
Performing Nucleic Acid Analysis

The Novex® TBE Gels are used to analyze DNA fragments including restriction digest, PCR products, Southern analysis, and primer analysis.  The Novex® TBE-Urea Gels are used for denaturing nucleic acid analysis and are suited for RNase Protection Assays, in-vitro transcription studies, RNA stability studies, and oligonucleotide purification.
 
Performing Gel Shift Assays

The Novex® 6% DNA Retardation Gels are used to perform gel shift assays.

Materials

TBE Gels

Novex ® polyacrylamide TBE Gels provide high-resolution analysis of restriction digests and PCR products. The TBE Gels give sharp, intense bands and provide separations of double-strand DNA fragments from 10-3000 base pairs.
 
Advantages

Using polyacrylamide gels for nucleic acid separation provides the following advantages over agarose gels:

  • High resolution and sensitivity
  • Lower background staining
  • Requires less sample concentration and volume
  • Efficient blotting
  • Easy to extract DNA from the gel and does not interfere with enzymatic reactions
  • Accurate and reproducible results

Materials Supplied by the User

You will need the following items:

  • DNA sample
  • Deionized water
  • Appropriate DNA markers
  • Hi-Density TBE Sample Buffer
  • TBE Running Buffer

Electrophoresis of Novex® Pre-Cast Gels

Instructions are provided below for electrophoresis of the Novex® Pre-Cast Gels using the XCell SureLock™ Mini-Cell.   If you are using any other mini-cell for electrophoresis, refer to the manufacturer’s recommendations.

Preparing Samples

The Novex® Hi-Density TBE Sample Buffer (5X) can be ordered from the above table.

  1. Prepare samples for TBE gels as described below:

 

ReagentAmount
Sample    x µl
Novex® Hi-Density TBE Sample Buffer (5X)    2 µl
Deionized Waterto 8 µl
Total Volume  10 µl

 

  1. Load the samples immediately on the gel.


Preparing Running Buffer

Novex® TBE Running Buffer (5X) is available from Thermo Fisher Scientific.
 

  1. Prepare 1000 ml of 1X TBE Running Buffer using Novex® TBE Buffer (5X) as follows:

 

Novex® TBE Running Buffer (5X)200 ml
Deionized Water800 ml
Total Volume1000 ml

 

  1. Mix thoroughly.  Use this buffer to fill the Upper and Lower Buffer Chamber of the XCell SureLock™ Mini-Cell for electrophoresis.

 
Electrophoresis Conditions
 
Migration of the Dye Fronts:  The size of the DNA fragments visualized at the dye fronts of the different TBE Gels is shown in the table below.

Gel Type
Dye Front*
 
Bromophenol Blue (dark blue)
Xylene Cyanol (blue green)
6% TBE Gel
65 bp
250 bp
8% TBE Gel
25 bp
220 bp
10% TBE Gel
35 bp
120 bp
20% TBE Gel
15 bp
50 bp
4-12% TBE Gel
35 bp
400 bp
4-20% TBE Gel
25 bp
300 bp

*accuracy is + 5 bp

Protocol using XCell SureLock™ Mini-Cell

Wear gloves and safety glasses when handling gels.
 
XCell SureLock™ Mini-Cell requires 200 ml for the Upper Buffer Chamber and 600 ml for the Lower Buffer Chamber.
 

  1. Remove the Novex® Pre-Cast Gel from the pouch.

  2. Rinse the gel cassette with deionized water.  Peel off the tape from the bottom of the cassette.

  3. In one smooth motion, gently pull the comb out of the cassette.

  4. Rinse the sample wells with the appropriate 1X SDS Running Buffer.  Invert the gel and shake the gel to remove the buffer.  Repeat two more times.  

  5. Orient the two gels in the Mini-Cell such that the notched “well” side of the cassette faces inwards toward the Buffer Core.  Seat the gels on the bottom of the Mini-Cell and lock into place with the Gel Tension Wedge.  Refer to the XCell SureLock™ Mini-Cell manual (IM-9003) for detailed instructions. 



  6. Fill the Upper Buffer Chamber with a small amount of the running buffer to check for tightness of seal.  If you detect a leak from Upper to the Lower Buffer Chamber, discard the buffer, reseal the chamber, and refill.

  7. Once the seal is tight, fill the Upper Buffer Chamber (inner) with the appropriate 1X running buffer.  The buffer level must exceed the level of the wells.

  8. Load an appropriate volume of sample at the desired protein concentration onto the gel.

  9. Load appropriate protein molecular weight markers.

  10. Fill the Lower Buffer Chamber with 600 ml of the appropriate 1X running buffer.

  11. Place the XCell SureLock™ Mini-Cell lid on the Buffer Core.  With the power on the power supply turned off, connect the electrode cords to the power supply [red to (+) jack, black to (-) jack].

 
Electrophoresis Conditions

Run your gels according to the following protocol:

Gel Type
Voltage
Expected Current*
Run Time
TBE Gels
200 V constant**
Start: 10-18 mA
End: 4-6 mA
30-90 minutes (dependent on gel type)
Run the gel until the bromophenol blue tracking dye reaches the bottom of the gel.
6% TBE-Urea Gels
180 V constant**
Start: 19 mA
End: 14 mA
50 minutes
Run the gel until the bromophenol blue tracking dye reaches the bottom of the gel.
10% TBE-Urea Gels
180 V constant**
Start: 15 mA
End: 8 mA
60 minutes
Run the gel until the bromophenol blue tracking dye reaches the bottom of the gel.
15 % TBE-Urea Gels
180 V constant**
Start: 13 mA
End: 6 mA
75 minutes
Run the gel until the bromophenol blue tracking dye reaches the bottom of the gel.
DNA Retardation Gels
100 V constant
Start: 12-15 mA
End: 6-15 mA
90 minutes
Run the gel until the bromophenol blue tracking dye reaches the bottom of the gel.

 *Expected start and end current values are stated for single gels.
**Voltages up to 250 V may be used to reduce the run time.

Removing the Gel after Electrophoresis
 

  1. After electrophoresis is complete, shut off the power, disconnect electrodes, and remove gel(s) from the XCell SureLock™ Mini-Cell.

  2. Separate each of the three bonded sides of the cassette by inserting the Gel Knife into the gap between the cassette’s two plates.  The notched (“well”) side of the cassette should face up.

  3. Push down gently on the knife handle to separate the plates.  Repeat on each side of the cassette until the plates are completely separated.

    Caution:   Use caution while inserting the gel knife between the two plates to avoid excessive pressure towards the gel.

  4. Carefully remove and discard the top plate, allowing the gel to remain on the bottom (slotted) plate.

  5. If staining, remove the gel from the plate by one of the methods:
  • Use the sharp edge of the gel knife to remove the bottom lip of the gel.  The gel knife should be at a 90° angle, perpendicular to the gel and the slotted half of the cassette.  Push down on the knife, and then repeat the motion across the gel to cut off the entire lip.  Hold the plate and gel over a container with the gel facing downward and use the knife to carefully loosen one lower corner of the gel and allow the gel to peel away from the plate.
  • Hold  the plate and gel over a container with the gel facing downward.  Gently push the gel knife through the slot in the cassette, until the gel peels away from the plate.  Cut the lip off of the gel after fixing, staining, but before drying.

 

  1. Fix and stain the gel.

Troubleshooting

Problem
Cause
Solution
Run taking longer time
Running buffer too dilute
Make fresh running buffer as described in this manual and avoid adjusting the pH of the 1X running buffer.
Low or no current during the run
Incomplete circuit
  • Remove the tape from the bottom of the cassette prior to electrophoresis.
  • Make sure the buffer covers the sample wells.
  • Check the wire connections on the buffer core to make sure the connections are intact.
Faint shadow or “ghost” band below the expected protein band
Ghost bands are caused due to a slight lifting of the gel from the cassette resulting in trickling of some sample beyond its normal migration point. Gel lifting off the cassette is caused due to:

  • Expired gels
  • Improper storage of gels
 
  • Avoid using expired gels. Use fresh gels
  • Store the gels at the appropriate temperature.
Streaking of proteins
  • Sample overload
  • Load the appropriate amount of protein as described.
 
  • High salt concentration in the sample
  • Decrease the salt concentration of your sample using dialysis or gel filtration
 
  • Sample precipitates
  • Increase the concentration of SDS in your sample if necessary, to maintain the solubility of the protein.
 
  • Contaminants such as membranes or DNA complexes in the sample
  • Centrifuge or clarify your sample to remove particulate contaminants
Bands in the outer lane of the gel are curving upwards
  • Concentrated buffer used
  • The pre-made buffers are supplied as concentrate. Dilute the buffers as described.
 
 
  • Expired gels used
  • Avoid using gels after the expiration date.
 
  • High voltage used
  • Electrophorese the gel using conditions described.
Bands in the outside lanes of the gel “smiling”
Expired gels used causing the acrylamide to break down in the gel
Avoid using gels after the expiration date. Use fresh gels.
Bands are running as U shape rather than a flat band
Samples are loaded on the gel and not electrophoresed immediately resulting in sample diffusion
Load samples on to the gel immediately before electrophoresis.
Bands appear to be “funneling” or getting narrower as they progress down the gel
Proteins are over-reduced causing the proteins to be negatively charged and repel each other.
Reduce the proteins using DTT or b-mercaptoethanol as described.
Dumbbell shaped bands after electrophoresis
Loading a large volume of sample causing incomplete stacking of the entire sample. This effect is intensified for larger proteins
Load the appropriate volume of sample per well as described. If your sample is too dilute, concentrate the sample using salt precipitation or ultrafiltration.
For TBE-Urea gels
High background and smeared bands or abnormal band shapes
  • RNase contamination
  • Always wear gloves and use sterile techniques to prevent RNase contamination.
 
  • Sample renatured
  • Heat the sample for 3 minutes at 70°C and keep the sample in ice to prevent renaturation. Proceed to electrophoresis immediately after loading.
 
  • Sample overloaded
  • Recommended DNA load is 0.16-0.33 µg/band.
 
  • Urea not completely flushed from the wells
  • Be sure to thoroughly flush urea out of the wells prior to loading the sample.
LT108