ELECTROPHORESIS OF DNA IN POLYACRYLAMIDE GELS

ELECTROPHORESIS OF DNA IN POLYACRYLAMIDE GELS

Gel Sizes
Small:             165 x 130 mm
Medium:         165 x 200 mm
Large:            165 x 260 mm

5% Analytical Gels

Reagent	<center><b><font>1 mm Small</font> </b> </center>	<center><b><font>1 mm Medium</font> </b> </center>	<center><b><font>1 mm Large</font> </b> </center>
10X TBE Buffer (ml)	<center><font>3</font> </center>	<center><font>3.5</font> </center>	<center><font>5</font> </center>
40% bis/acrylamide (ml)	<center><font>3.75</font> </center>	<center><font>4.4</font> </center>	<center><font>6.2</font> </center>
Water (ml)	<center><font>17.25</font> </center>	<center><font>20.1</font> </center>	<center><font>28.8</font> </center>
50% Glycerol (ml)	<center><font>6</font> </center>	<center><font>7</font> </center>	<center><font>10</font> </center>
APS (mg)	<center><font>34</font> </center>	<center><font>40</font> </center>	<center><font>57</font> </center>
TEMED (ul)	<center><font>12.8</font> </center>	<center><font>15</font> </center>	<center><font>21.4</font> </center>
Total Volume (ml)	<center><font>30</font> </center>	<center><font>35</font> </center>	<center><font>50</font> </center>

With other concentrations of a bis/acrylamide stock substitute:


25% bis/acrylamide (ml)	6.0	7.0	10.0
Water (ml)	15.0	18.5	25.0




30% bis/acrylamide (ml)	5.0	5.8	8.3
Water (ml)	16.0	18.7	26.7




50% bis/acrylamide (ml)	3.0	3.5	5.0
Water (ml)	18.0	21.0	30.0

5% BAC Cross-linked Polyacrylamide Gels

Reagent	1 mm Medium	1 mm Long	4 mm Small	4 mm Medium
10x TBE Buffer (ml)	<center><font>8</font> </center>	<center><font>10</font> </center>	<center><font>16</font> </center>	<center><font>20</font> </center>
Water (ml)	<center><font>14</font> </center>	<center><font>17.5</font> </center>	<center><font>28</font> </center>	<center><font>35</font> </center>
50% Glycerol (ml)	<center><font>8</font> </center>	<center><font>10</font> </center>	<center><font>16</font> </center>	<center><font>20</font> </center>
20% bac/acrylamide (ml)	<center><font>10</font> </center>	<center><font>12.5</font> </center>	<center><font>20</font> </center>	<center><font>25</font> </center>
APS (mg)	<center><font>16</font> </center>	<center><font>20</font> </center>	<center><font>32</font> </center>	<center><font>40</font> </center>
TEMED (ml)	<center><font>200</font> </center>	<center><font>250</font> </center>	<center><font>400</font> </center>	<center><font>500</font> </center>
Total Volume (ml)	<center><font>40</font> </center>	<center><font>50</font> </center>	<center><font>80</font> </center>	<center><font>100</font> </center>

Pre-run gels for 30 minutes at 80V. Rinse the wells with running buffer. Load and electrophorese samples at 80V until dyes separate, then boost to up to 150 V. Medium sized polyacrylamide gels can be run overnight at 55-60 V to see all phi-X/Hae III cut molecular weight standards on the gel.


7.5% Mini (10 x 8.2 cm) Polyacrylamide Gels

10 ml 7.5% Polyacrylamide Gel Solution

65 µl 10% (w/v) APS

12.5 µl TEMED


SEQUENCING GELS (8% Polyacrylamide, 8 M Urea)
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Reagent	40 cm gel	80 cm gel	1 m gel
Urea (g)	<center><font>48</font> </center>	<center><font>86.5</font> </center>	<center><font>120</font> </center>
Water (ml)	<center><font>37</font> </center>	<center><font>66.5</font> </center>	<center><font>92.4</font> </center>
50% bis/acrylamide (ml)	<center><font>16</font> </center>	<center><font>28.8</font> </center>	<center><font>40</font> </center>
10x TBE Buffer (ml)	<center><font>10</font> </center>	<center><font>18</font> </center>	<center><font>25</font> </center>
10% (w/v) APS (ml)	<center><font>0.668</font> </center>	<center><font>1.2</font> </center>	<center><font>1.67</font> </center>
TEMED (ml)	<center><font>50</font> </center>	<center><font>85</font> </center>	<center><font>118</font> </center>
Total Volume (ml)	<center><font>100</font> </center>	<center><font>180</font> </center>	<center><font>250</font> </center>

Pre-run sequencing gels at 1800 V for 30 minutes.

With other concentrations of a bis/acrylamide stock substitute:

30% bis/acrylamide(ml)	<center><font> </font> </center>	<center><font>48</font> </center>	<center><font>66.7</font> </center>
Water (ml)	<center><font> </font> </center>	<center><font>47.3</font> </center>	<center><font>65.7</font> </center>

40% bis/acrylamide (ml)	<center><font> </font> </center>	<center><font>36</font> </center>	<center><font>50</font> </center>
Water (ml)	<center><font> </font> </center>	<center><font>59.3</font> </center>	<center><font>82.4</font> </center>

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<center><b><u><font>RECIPES</font> </u> </b> </center>

10X TBE (1M Tris, 1M Boric Acid, 20mM EDTA, pH 8.3)

242.2 g Tris

123.66 g boric acid

14.89 g EDTA

Adjust pH to 8.3. QS to 2 liters. Autoclave. (Biotechniques 10:182, 1991 claims that filtering up to a 20x TBE solution through 0.2 - 0.45µ cellulose acetate or cellulose nitrate filters prevents formation of precipitants during long-term storage. The solution may be reautocalved to dissolve precipitates that form.)

50% Glycerol

25 ml 100% glycerol

25 ml water

Autoclave. Concentrated glycerol is quite viscous. Be sure all of it has been transferred from the stock bottle.

30% Acrylamide Solution (0.8% bis)

60 g acrylamide

1.6 g bis

Heat to dissolve in approximately 100 ml of water. QS to 200 ml with water and filter with Whatman #1 filter paper into a foil wrapped bottle.

40% Acrylamide Solution (19:1 acrylamide:bis)

80 g acrylamide

4.21 g bis

Heat to dissolve in approximately 100 ml of water. QS to 200 ml with water and filter with Whatman #1 filter paper into a foil wrapped bottle.

50% Acrylamide Solution (19:1 acrylamide:bis)

237.5 g (97%) acrylamide

12.5 g bis

Dissolve in 250 ml hot water. Filter to remove debris and wrap in foil.

20% Bac-Acrylamide Solution     WARNING: wear gloves

100 ml solution

18.98 g acrylamide

1.02 g BAC (N',N'-bis-acrylylcystamine)

Heat approximately 75 ml of water to almost boiling. Add acrylamide and cover with a watch glass. Mix briefly to dissolve and reheat to near boiling. Add BAC. QS to 100 ml with water. Do not autoclave. Filter sterlize with Nalgene 0.45 or 0.2 micron filter and store in foil wrapped bottle.

7.5% Polyacrylamide Gel Solution

40% Bis/Acrylamide Stock Solution	18.75 ml	37.5 ml
10x TBE buffer	10 ml	20 ml
50% glycerol	20 ml	40 ml
Water	51.25 ml	102.5 ml
TOTAL VOLUME	100 ml	200 ml