Griffitts:Restriction digest: Difference between revisions
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''Adapted from New England BioLabs 2005-06 Catalog and Technical Reference.'' | |||
==Double Digests== | |||
{| border="1" cellpadding="5" cellspacing="0" class="wikitable sortable" | |||
|+'''Suggested Buffers''' | |||
|- | |||
! width="30" style="background:#efefef;" | Enzyme | |||
! width="30" style="background:#efefef;" | ''Avr'' II | |||
! width="30" style="background:#efefef;" | ''Bam'' HI | |||
! width="30" style="background:#efefef;" | ''Bgl'' II | |||
! width="30" style="background:#efefef;" | ''Bst'' XI | |||
! width="30" style="background:#efefef;" | ''Eco'' RI | |||
! width="30" style="background:#efefef;" | ''Eco'' RV | |||
! width="30" style="background:#efefef;" | ''Hind'' III | |||
! width="30" style="background:#efefef;" | ''Kpn'' I | |||
! width="30" style="background:#efefef;" | ''Nde'' I | |||
! width="30" style="background:#efefef;" | ''Pst'' I | |||
! width="30" style="background:#efefef;" | ''Sac'' I | |||
! width="30" style="background:#efefef;" | ''Sal'' I | |||
! width="30" style="background:#efefef;" | ''Sma'' I | |||
! width="30" style="background:#efefef;" | ''Spe'' I | |||
! width="30" style="background:#efefef;" | ''Sph'' I | |||
! width="30" style="background:#efefef;" | ''Xba'' I | |||
! width="30" style="background:#efefef;" | ''Xho'' I | |||
|- | |||
| ''Bam'' HI | |||
| seq | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
|- | |||
| ''Bgl'' II | |||
| 3 | |||
| ''Bam'' HI | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
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<br><br> | <br><br> | ||
''Adapted from New England BioLabs 2005-06 Catalog and Technical Reference.'' | ''Adapted from New England BioLabs 2005-06 Catalog and Technical Reference.'' |
Revision as of 15:31, 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 |
---|---|---|---|---|---|---|---|---|---|---|
Avr II | c/ctagg | ctag | 100 | 100 | 50 | 100 | None | 37°C | No | |
Bam HI | 1 μL | g/gatcc | gatc | 75 | 100 | 50 | 75 | Bam HI | 37°C | Yes |
Bgl II | a/gatct | gatc | 10 | 75 | 100 | 10 | None | 37°C | No | |
Bst XI | ccannnnn/ntgg | nnnn | 25 | 100 | 100 | 50 | None | 55°C | No | |
Eco RI | g/aattc | aatt | 100 | 100 | 100 | 100 | Eco RI | 37°C | No | |
Eco RV | gat/atc | none | 50 | 75 | 100 | 50 | None | 37°C | Yes | |
Hind III | a/agctt | agct | 50 | 100 | 10 | 50 | None | 37°C | No | |
Kpn I | ggtac/c | gtac | 100 | 75 | 0 | 75 | None | 37°C | Yes | |
Nde I | ca/tatg | ta | 75 | 100 | 75 | 100 | None | 37°C | No | |
Pst I | ctgca/g | tgca | 75 | 75 | 100 | 50 | None | 37°C | Yes | |
Sac I | gagct/c | agct | 100 | 50 | 10 | 100 | None | 37°C | Yes | |
Sal I | g/tcgac | tcga | 0 | 0 | 100 | 0 | None | 37°C | Yes | |
Sma I | ccc/ggg | none | 0 | 0 | 0 | 100 | None | 25°C | No | |
Spe I | a/ctagt | ctag | 75 | 100 | 25 | 75 | None | 37°C | Yes | |
Sph I | gcatg/c | catg | 100 | 100 | 50 | 100 | None | 37°C | No | |
Xba I | 1.5 μL | t/ctaga | ctag | 0 | 100 | 75 | 75 | None | 37°C | Yes |
Xho I | c/tcgag | tcga | 75 | 100 | 100 | 100 | None | 37°C | Yes |
Adapted from New England BioLabs 2005-06 Catalog and Technical Reference.
Double Digests
Enzyme | Avr II | Bam HI | Bgl II | Bst XI | Eco RI | Eco RV | Hind III | Kpn I | Nde I | Pst I | Sac I | Sal I | Sma I | Spe I | Sph I | Xba I | Xho I |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Bam HI | seq | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - |
Bgl II | 3 | Bam HI | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - |
Adapted from New England BioLabs 2005-06 Catalog and Technical Reference.