DNA sequencing gels

Tuesday, November 18, 2003

Description
sequencing gels

Procedure
1) Clean the glass plates extensively with detergent and water, tap water, distilled water and finally ethanol. Wipe dry with a clean paper towel.

2) Siliconize the smaller of the two plates using the 4% solution of dichlorodimethylsilane in hexane. The solution should be spread evenly over the plate and allowed to dry before being repeated. Once dry, the plate should be washed with 100% ethanol and again wiped dry using a clean paper towel.

3) Gel plates are then assembled as described in the manufacturers instructions using two 0.25 -1mm wedge spacers.

Polyacrylamide sequencing mix for use in the gels was stored at 4C in a dark bottle.

4) 35ml of the acrylamide mix is used to first plug the bottom of the gel. Chill the acrylamide on ice and add 150ul 25% AMPS and 150ul TEMED. Mix by swirling and then poured briskly into the gel mould. The quantities of AMPS and TEMED may have to be esti mated empirically to cause setting in approx. 5 minutes.

5) Once the plug has set, 85ml of acrylamide is then used to form the main gel itself. To the acrylamide (chilled on ice beforehand), add 110ul 25% AMPS and 110ul TEMED. The solutions are mixed thoroughly, placed into a 50ml syringe and injected, carefull y, between the glass plates. In order to facilitate ease of pouring, the glass plates were inclined at an angle of approximately 10 to the horizontal in a large developing tray to prevent spills. Again, the quantities of AMPS and TEMED used may need to be varied in order to give polymerisation in approx. 30 minutes - this may be especially critical if the ambient temperature is abnormally warm.

N.B: It is critical to chill the acrylamide for the main gel in order to prevent polymerisation while the gel is being poured. You may also need to adjust the AMPS/TEMED quantities used. You should aim to have the plug set in ~5 mins and the main g el after ~30 mins.

6) Immediately after the gel is poured, a flat 0.25mm spacer (or reversed shark tooth comb) should be placed into the acrylamide on the gel top such that it intrudes into the gel by approximately 10mm. This allows the formation of a flat gel surface essen tial to the effective use of the shark tooth combs during electrophoresis. Clamp large bulldog clips across the top of the gel plates during gel polymerisation to ensure a leak-free fit of the combs. Allowed to polymerise for 1 hour at room temperature an d then use directly or store overnight at 4C, tightly wrapped in clingfilm to prevent dehydration of the gel.

7) Remove the gel former and pre-electrophorese the gel at 1800V to heat the gel and running buffer to the required operating temperature (55C) prior to the loading of the samples. Running buffer is 1 x TBE.

8) Insert sharks tooth combs such that the tips protrude approximately 0.5mm into the gel surface.

9) Thoroughly wash the wells immediately prior to the loading of the samples with running buffer to remove any urea which leaches from the gel.

10) Sequencing reaction mixtures, containing loading buffer, should be boiled for 2-3 minutes to denature any secondary structure and loaded into the wells (3ul/well), in the order G, A, T, C.

11) Electrophorese at 1800V (preferably 75W constant power) until the xylene cyanol dye front is approximately 5 cm from the bottom of the gel. Monitor the gel temperature to ensure it stays at 60C or below (preferably 50-55C).

N.B: Allowing the gel temperature to exceed 60C for extended periods of time will cause the hydrolysis of urea in the gel.

12) After electrophoresis is complete, combs should be removed and the small siliconized glass plate gently removed from the remaining plate. The large plate, with the gel still attached, is then immersed in a fixative solution containing 10% acetic acid, 10% methanol for approximately 15-20 minutes. This process is used to remove urea from the gel.

13) Transfer the gel to a large sheet of Whatman 3MM paper and dry on a vacuum gel drier at 85C for 75 minutes prior to autoradiography.

Recipes
A standard detergent
2% dichlorodimethyl silane in hexane
Absolute ethanol
6% sequencing acrylamide (5.7% acrylamide, 0.3% bisacrylamide, 48% urea, 1x TBE)
25% AMPS (freshly made)
TEMED

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DNA sequencing gels