Griffitts:Restriction digest: Difference between revisions
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! width="60" style="background:#efefef;" | NEBuffer3 | ! width="60" style="background:#efefef;" | NEBuffer3 | ||
! width="60" style="background:#efefef;" | NEBuffer4 | ! width="60" style="background:#efefef;" | NEBuffer4 | ||
! width="60" style="background:#efefef;" | Other Buffer | |||
! width="60" style="background:#efefef;" | T° | |||
! width="60" style="background:#efefef;" | BSA | |||
|- | |- | ||
| ''Avr''II | | ''Avr''II | ||
Line 41: | Line 44: | ||
| 50 | | 50 | ||
| 100 | | 100 | ||
| None | |||
| 37°C | |||
| No | |||
|- | |- | ||
| ''Bam''HI | | ''Bam''HI | ||
Line 50: | Line 56: | ||
| 50 | | 50 | ||
| 75 | | 75 | ||
| ''Bam''HI | |||
| 37°C | |||
| Yes | |||
|- | |- | ||
| ''Bgl''II | | ''Bgl''II | ||
Line 59: | Line 68: | ||
| '''100''' | | '''100''' | ||
| 10 | | 10 | ||
| None | |||
| 37°C | |||
| No | |||
|- | |- | ||
| ''Bst''XI | | ''Bst''XI | ||
Line 68: | Line 80: | ||
| '''100''' | | '''100''' | ||
| 50 | | 50 | ||
| None | |||
| 55°C | |||
| No | |||
|- | |- | ||
| ''Eco''RI | | ''Eco''RI | ||
Line 77: | Line 92: | ||
| 100 | | 100 | ||
| 100 | | 100 | ||
| ''Eco''RI | |||
| 37°C | |||
| No | |||
|- | |- | ||
| ''Eco''RV | | ''Eco''RV | ||
Line 86: | Line 104: | ||
| '''100''' | | '''100''' | ||
| 50 | | 50 | ||
| None | |||
| 37°C | |||
| Yes | |||
|- | |- | ||
| ''Hind''III | | ''Hind''III | ||
Line 95: | Line 116: | ||
| 10 | | 10 | ||
| 50 | | 50 | ||
| None | |||
| 37°C | |||
| No | |||
|- | |- | ||
| ''Kpn''I | | ''Kpn''I | ||
Line 104: | Line 128: | ||
| 0 | | 0 | ||
| 75 | | 75 | ||
| None | |||
| 37°C | |||
| Yes | |||
|- | |- | ||
| ''Nde''I | | ''Nde''I | ||
Line 113: | Line 140: | ||
| 75 | | 75 | ||
| '''100''' | | '''100''' | ||
| None | |||
| 37°C | |||
| No | |||
|- | |- | ||
| ''Pst''I | | ''Pst''I | ||
Line 122: | Line 152: | ||
| '''100''' | | '''100''' | ||
| 50 | | 50 | ||
| None | |||
| 37°C | |||
| Yes | |||
|- | |- | ||
| ''Sac''I | | ''Sac''I | ||
Line 131: | Line 164: | ||
| 10 | | 10 | ||
| 100 | | 100 | ||
| None | |||
| 37°C | |||
| Yes | |||
|- | |- | ||
| ''Sal''I | | ''Sal''I | ||
Line 140: | Line 176: | ||
| '''100''' | | '''100''' | ||
| 0 | | 0 | ||
| None | |||
| 37°C | |||
| Yes | |||
|- | |- | ||
| ''Sma''I | | ''Sma''I | ||
Line 149: | Line 188: | ||
| 0 | | 0 | ||
| '''100''' | | '''100''' | ||
| None | |||
| 25°C | |||
| No | |||
|- | |- | ||
| ''Spe''I | | ''Spe''I | ||
Line 158: | Line 200: | ||
| 25 | | 25 | ||
| 75 | | 75 | ||
| None | |||
| 37°C | |||
| Yes | |||
|- | |- | ||
| ''Sph''I | | ''Sph''I | ||
Line 167: | Line 212: | ||
| 50 | | 50 | ||
| 100 | | 100 | ||
| None | |||
| 37°C | |||
| No | |||
|- | |- | ||
| ''Xba''I | | ''Xba''I | ||
Line 176: | Line 224: | ||
| 75 | | 75 | ||
| 75 | | 75 | ||
| None | |||
| 37°C | |||
| Yes | |||
|- | |- | ||
| ''Xho''I | | ''Xho''I | ||
Line 185: | Line 236: | ||
| 100 | | 100 | ||
| 100 | | 100 | ||
| None | |||
| 37°C | |||
| Yes | |||
|- | |- | ||
|} | |} |
Revision as of 15:02, 8 November 2007
Restriction Digest
Materials
- ddH20
- DNA sample on ice
- Appropriate 10X buffer on ice
- 10X BSA on ice
- Restriction enzyme(s) (see below) on ice
- CIP (keep in freezer until needed)
Recipe
- 12-20 μL DNA depending on concentration
- 4.3 μL appropriate 10X buffer
- 4.0 μL 10X BSA
- Restriction enzyme(s) (see specific enzymes below for exact amounts)
- Don't immerse the tip, draw from the surface to avoid excess enzyme
- Add ddH20 to bring the final volume to 40 μL
Procedure
- Combine ingredients for recipe
- Incubate at 37°C for 2.5 to 8 hours
- If preparing a ligation, add 0.5 μL CIP to the vector 30 minutes prior to ending the reaction
- When the reaction is complete you can store your samples in the -20°C freezer
Enzymes
Enzyme | Amount | Target | Overhang | NEBuffer1 | NEBuffer2 | NEBuffer3 | NEBuffer4 | Other Buffer | T° | BSA |
---|---|---|---|---|---|---|---|---|---|---|
AvrII | c/ctagg | ctag | 100 | 100 | 50 | 100 | None | 37°C | No | |
BamHI | 1 μL | g/gatcc | gatc | 75 | 100 | 50 | 75 | BamHI | 37°C | Yes |
BglII | a/gatct | gatc | 10 | 75 | 100 | 10 | None | 37°C | No | |
BstXI | ccannnnn/ntgg | nnnn | 25 | 100 | 100 | 50 | None | 55°C | No | |
EcoRI | g/aattc | aatt | 100 | 100 | 100 | 100 | EcoRI | 37°C | No | |
EcoRV | gat/atc | none | 50 | 75 | 100 | 50 | None | 37°C | Yes | |
HindIII | a/agctt | agct | 50 | 100 | 10 | 50 | None | 37°C | No | |
KpnI | ggtac/c | gtac | 100 | 75 | 0 | 75 | None | 37°C | Yes | |
NdeI | ca/tatg | ta | 75 | 100 | 75 | 100 | None | 37°C | No | |
PstI | ctgca/g | tgca | 75 | 75 | 100 | 50 | None | 37°C | Yes | |
SacI | gagct/c | agct | 100 | 50 | 10 | 100 | None | 37°C | Yes | |
SalI | g/tcgac | tcga | 0 | 0 | 100 | 0 | None | 37°C | Yes | |
SmaI | ccc/ggg | none | 0 | 0 | 0 | 100 | None | 25°C | No | |
SpeI | a/ctagt | ctag | 75 | 100 | 25 | 75 | None | 37°C | Yes | |
SphI | gcatg/c | catg | 100 | 100 | 50 | 100 | None | 37°C | No | |
XbaI | 1.5 μL | t/ctaga | ctag | 0 | 100 | 75 | 75 | None | 37°C | Yes |
XhoI | c/tcgag | tcga | 75 | 100 | 100 | 100 | None | 37°C | Yes |