Griffitts:Restriction digest
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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 |
---|---|---|---|---|---|---|---|
AvrII | c/ctagg | ctag | 100 | 100 | 50 | 100 | |
BamHI | 1 μL | g/gatcc | gatc | 75 | 100 | 50 | 75 |
BglII | a/gatct | gatc | 10 | 75 | 100 | 10 | |
BstXI | ccannnnn/ntgg | nnnn | 25 | 100 | 100 | 50 | |
EcoRI | g/aattc | aatt | 100 | 100 | 100 | 100 | |
EcoRV | gat/atc | none | 50 | 75 | 100 | 50 | |
HindIII | a/agctt | agct | 50 | 100 | 10 | 50 | |
KpnI | ggtac/c | gtac | 100 | 75 | 0 | 75 | |
NdeI | ca/tatg | ta | 75 | 100 | 75 | 100 | |
PstI | ctgca/g | tgca | 75 | 75 | 100 | 50 | |
SacI | gagct/c | agct | 100 | 50 | 10 | 100 | |
SalI | g/tcgac | tcga | 0 | 0 | 100 | 0 | |
SmaI | ccc/ggg | none | 0 | 0 | 0 | 100 | |
SpeI | a/ctagt | ctag | 75 | 100 | 25 | 75 | |
SphI | gcatg/c | catg | 100 | 100 | 50 | 100 | |
XbaI | 1.5 μL | t/ctaga | ctag | 0 | 100 | 75 | 75 |
XhoI | c/tcgag | tcga | 75 | 100 | 100 | 100 |