MarcantonioLabProtocolManual——3

Sequencing Gel

Preparing and Running a Sequencing Gel (6% Polyacrylamide/Urea) 

A. Preparation of Gel Solution 

1) Weigh out 50 g of Urea into a clean 250 ml beaker containing a stir bar. 
2) Add to the beaker:

• DD-H2O 40 ml

• 40% acrylamide stock (19:1 Acryl/Bis) 15 ml

• 20x TBE 5 ml

3) Dissolve on a stirrer. 

B. Assembling the Gel Sandwich 

1) Clean two glass plates, one small and one large, by THOROUGHLY scrubbing with a non-abrasive detergent and brush. Rinse THOROUGHLY with water and allow to air dry. Be sure to remove all traces of ink, soap, tape, dirt, dust, sweat, lunch, and any other debris that may settle on the plates. Any such contamination may result in extreme difficulty when pouring the gel. 
2) When plates are nearly dry, wipe with DD-H2O and dry, then wipe with ethanol and let dry. 
3) Siliconize one surface of the larger plate by wiping its surface with Sigmacote applied to a Kimwipe. This procedure should be performed in a fume hood to avoid contact with the Sigmacote vapors.

Siliconizing one plate helps the completed sequencing gel adhere preferentially to the other plate when disassembling the sandwich. 

4) Immediately before assembling plates, rinse them with ethanol and dry with a Kimwipe. 
5) Place the larger plate, siliconized side up, on lab paper. Place the vinyl side spacers along the long edge of the plate with the foam block face up. Carefully place the smaller plate over this so that the lower edges of the two plates meet and the foam spacer block is snug against the top of the smaller plate.. 
6) Tape the edges of the plates to seal the sandwich. First tape the bottom edges, then the sides.

The tape should be firmly pulled and pressed against each surface of the glass plates in order to accomplish a leak-free seal. Each edge should be sealed thoroughly with two strips of tape. Special attention should be paid to sealing the lower corners of the sandwich. 

C. Pouring the Gel 

1) Immediately before pouring the gel, filter the gel solution through a disposable Nalgene unit and pour into a fresh, clean 250 ml beaker. 
2) Add the following reagents to the filtered gel solution:

• TEMED 15 ul

• 10% Ammonium Persulfate 1 ml

Following addition of the APS use the pipet tip to stir the solution. These reagents catalyze the polymerization of the gel solution. Depending on the precise environmental conditions, you have approximately 10 minutes to pour the gel after the addition of the catalysts. 

3) Holding the plates at a 45 degree angle so that the gel will flow down one of the side spacers, carefully inject the gel between the glass plates using a 50 ml syringe, maintaining an even flow to avoid the formation of air bubbles. If air bubbles form, move the plates to a more vertical position and tap the glass plates to work the bubbles out. 
4) Pour gel solution until there is a reservoir of liquid extruding from between the plates when they are resting at a 5 degree angle. 
5) Insert the flat edge of a sharkstooth comb between the plates to an approximate depth of 2-3 mm beneath the edge of the short plate. Insert the other comb likewise, making the edges of the two combs meet evenly. 
6) Place a spring clip on both sides of the sandwich over the combs to keep them in place. 
7) Leave the plates in this position to polymerize. 
8) If the gel is going to be used the same day then skip to the next section of this protocol, D. Setting Up the Gel. If the gel is to used at a later time, then remove the spring clips, soak several paper towels in water and wrap them around the top part of the gel to prevent it from drying out. Then, wrap the entire top half of the sandwich in plastic wrap and reapply the spring clips. 

D. Setting Up the Gel 

1) Remove the spring clips from the top of the gel, as well as any paper towels and plastic wrap, if used. Remove the tape from the lower edge of the glass plates by cutting with a razor blade. 
2) Position the gel sandwich so that the shorter plate faces the apparatus and the foam blocks form a seal with the gasket. Do not yet tighten the screw knobs. 
3) Carefully remove the sharkstooth combs from the gel, keeping track of which comb went in which side of the gel. Rinse the combs with water and place them between the plates so that the teeth just make contact with the gel surface. Clamp the combs in place using thumb screws.

If done properly, a slight indentation will be evident where the teeth touch the gel surface. Do not puncture the gel surface or tear it by moving the inserted combs laterally. 

4) Gradually tighten the screw knobs, in an alternating manner, to complete the seal. Be careful not to overtighten the screws as this may damage the plates. 
5) Close the upper buffer drain valve and fill the upper and lower reservoirs with 500 ml of TBE electrophoresis buffer in each. 
6) Close the upper and lower safety lids and attatch the apparatus to the power supply. (Red lead on bottom, black lead on top) Gel should be pre-electrophoresed at 30 watts, constant power, for 15-30 minutes before loading samples, although this is not necessary. 

E. Sample Application 

1) If the sequencing reactions were just completed, then the samples must be heated to 75 degrees for 2 minutes prior to loading. If they were stored at -20 before running the gel, then they should be allowed to warm up for a few minutes prior to loading. 
2) Immediately prior to loading the samples, the wells must be washed out to remove the urea which leeches into them from the gel. Use a syringe with a bent needle to rinse the wells with buffer. If the wells are not rinsed properly, then the sample will not form a tight band as it migrates through the gel and band resolution on the autoradiograph will suffer. 
3) Using the Eppendorf Ultra-Micro pipettor, load 3 ul of sample into each well, loading in the order G-A-T-C. 
4) Close the upper safety lid, reattatch the black lead and switch on the power supply to provide 60 watts of constant power. 

F. Disassembling the Gel 

1) Turn off the power supply and disconnect the leads from the apparatus. 
2) Open the upper chamber drain valve to release the contents of the upper buffer chamber into the rear portion of the lower buffer chamber. 
3) Loosen the screw knobs and remove the gel sandwich from the apparatus. The buffer in the front of the lower buffer chamber contains radioactive nucleotides and care should be taken not to drip this on unprotected surfaces when moving the gel sandwich. 
4) The buffer in the front of the lower buffer chamber should be carefully poured into a large beaker to be stored in the radiation hood for future disposal. 
5) Remove the tape from the gel sandwich and slide out the side spacers and the combs. 
6) Place the sandwich on a protected lab bench with the small plate on top. Being extremely careful not to tear the gel, carefully pry the plates apart using a spatula. The gel should stick preferentially to the large plate, which has not been siliconized. However, if it decides to adhere to the small plate instead, then go with the flow. It doesn't matter which plate it sticks to as long as it is all on one plate! 
7) Cut a piece of Whatmans #3 blotting paper to approximately the size of the gel and press it firmly and evenly onto the surface of the gel, especially at the corners and edges. 
8) Carefully lift a corner of the paper to see if the gel is adhering to it. If so , then continue lifting the paper away from that corner to remove the gel from the glass plate.

If the gel is not sticking to that corner, then try lifting at another corner or edge. If there is still difficulty, then wet the paper a bit using a squeeze bottle and try again. 

G. Exposing the Gel 

1) Place the gel on the gel dryer, backing paper down. Cover the surface of the gel with a layer of plastic wrap and cover with the rubber seal. 
2) After making sure that the vacuum traps are empty and the second flask is immersed in a slurry of dry ice and ethanol, apply a vacuum and turn on the dryer. It should be set at 80 degrees for 40 minutes. 
3) When the gel is finished drying, first break the vacuum by lifting the rubber seal and turning the pump off. 
4) Remove the plastic wrap from the gel and expose it directly to XAR film in an autoradiograph cassette overnight at -80 degrees. No intensifying screen is needed. In fact, it can decrease the resolution of the fragments on the gel. 
5) Develop the film in the X-OMAT Film Processor. If necessary, re-expose for a longer period to enhance the readability. 

Transformation

Transformation of HB-101 Competent Cells 

1) Thaw tubes of competent cells on ice, occasionally inverting gently to mix. 
2) Pre-cool Falcon 2059 tubes on ice. 
3) Aliquot 30 ul cells into each tube. 
4) Add up to 10 ul DNA in TE or ligation buffer and place on ice for 10 minutes. This allows DNA to attatch to cells. 
5) Place in 42 degree water bath for 1 minute. This treatment shocks the cell membranes to allow take-up of the DNA. 
6) Add 0.5 ml of L2 media and shake at 37 degrees for up to 1-2 hours. Remember: Do not use media containing selection agent, usually ampicillin, as this is the period in which antibiotic resistance develops. 
7) Pipet cells onto L2 plates containing selective agent and spread using a sterile glass spreader (a bent Pasteur pipet). 
8) Wait until all liquid has been absorbed into plate, then incubate overnight at 37 degrees.