DNA Sequencing Gel
A. STOCK SOLUTIONS:
10 X NNB: Gel Fix:
162 g Tris base 10% Methanol
27.5 g Boric acid 10% Acetic Acid
9.3 g EDTA-Na2 2% Glycerol
870 ml Water
B. GEL:
BOTTOM MIX:
------------------------------------------------------------
Small Gel | Big Gel (BRL)
------------------------------------------------------------
Urea 4,8 g | 9,6 g 8 M
Glycerol 1,22 ml | 2,44 ml 10 %
Acrylamide 50%-Bis 2,5% 1,60 ml | 3,20 ml 8 %
NNB 10X 2,50 ml | 5,00 ml 2,5 X
Water 1,75 ml | 3,50 ml -
|
10% Persulfate 35 ul | 70 ul
TEMED 8 ul | 16 ul
-------------------------------------------------------------
TOTAL 10 ml | 20 ml
-------------------------------------------------------------
============================================================================
TOP MIX:
------------------------------------------------------------
Small Gel | Big Gel (BRL)
------------------------------------------------------------
Urea 19,2 g | 28,8 g 8 M
Acrtlamide 50%-Bis 2,5% 6,4 ml | 9,6 ml 8 %
NNB 10X 2,0 ml | 3,0 ml 0,5 %
Water 16,8 ml | 25,2 ml -
|
10% Persulfate 140 ul | 210 ul
TEMED 32 ul | 48 ul
-------------------------------------------------------------
TOTAL 40 ml | 60 ml
-------------------------------------------------------------
C. POURING:
01. Take 6 ml of Top Mix in a 25 ml pipet
02. Take 6 ml of Bottom Mix in the same pipet
03. Suck 1-2 bubles and let them pass through the solutions
04. Apply in the sequence plate
05. Complete with Top Mix
06. Inclinate the plate (approx. 15o)
07. Put the inverted comb (teeth upwards)
08. Secure the top with black clamps
09. Polymerize for at least 1 h
10. Take the clamps out, put a wet piece of Whatman 3MM a wrap with
Saran Wrap. Put the clamps back
11. Let it stand one night (that makes the gradient even)
D. RUNNING:
01. Make 1 liter of 1 x NNB
02. Take the tapes (or clamps) out of the plate
03. Take the comb out (with care!)
04. Fill the single well with 1 x NNB
05. Wash the outside of the glass plates with dd-water
06. Put the comb in the well with the teeth downwards, softly touching
the surface (DO NOT penetrate the gel!)
07. Wash the wells with 1 x NNB using a syringe (to remove excess of urea)
08. Heat samples at 85 oC for 3 min
09. Put the samples immediatelly in a water-ice mixture
10. Apply 3.5 ul of each sample and run at 30-45 watts for approx. 4h (TIL THE
XYLENE CYANOL BLUE RUNS OUT OF THE GEL)
11. Freeze the the reamining samples
12. If you are going to make only one run go to step [13]. If not turn the power supply
off, wash the wells again (see item 07) and apply the other half of the samples.
Run for 2h at the same current (TILL THE BROMOPHENOL BLUE RUNS OUT OF THE GEL)
13. Take the gel out, fix, dry and expose.