Wittrup: Plasmid miniprep: Difference between revisions

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Note: If the strain has high carbohydrate content or high nuclease activity (such as an endA+ strain) wash with 0.5 μL of Buffer PB before PE wash.
Note: If the strain has high carbohydrate content or high nuclease activity (such as an endA+ strain) wash with 0.5 μL of Buffer PB before PE wash.
'''Restriction Digest'''
''Materials''
DNA
Restriction enzyme(s)
Compatible buffer
37˚C water bath
''General Protocol''
The specific protocol can vary depending on the particular restriction enzyme.  The amount of enzyme needed (per μg of DNA) and the optimal temperature are generally available on the product specification sheet and/or the website.  The New England Biolabs website ([http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/default.asp]) provides useful information on a variety of issues relating to DNA digestion.
1.  Add ddH2O to tube (often 20-50 μL total volume).
2.  Add restriction digest buffer to tube.  Mix well (often provided as 10x concentrate).
3.  Add BSA (bovine serum albumin) if necessary (some enzymes require BSA for optimal activity).
4.  Add DNA to tube.  Mix well.
5.  Add restriction enzyme to tube.  Mix well.
6.  Incubate at appropriate temperature for required time.
7.  Heat inactivate the enzyme if desired and if possible.
''Specific Example''
1.  Add 16.6 μL of ddH2O to a microcentrifuge tube.
2.  Add 2 μL of 10x NEBuffer 2.  Mix well.
3.  Add 0.2 μL of 100x BSA.  Mix well.
4.  Add 1 μL of DNA (0.5 μg/μL).  Mix well.
5.  Add 0.2 μL of NheI (10 U/μL).  Mix well.
6.  Incubate at 37ºC for 2 h.
7.  Incubate at 65º for 20 min. to heat inactivate.

Revision as of 14:37, 6 March 2006

QIAprep Spin Miniprep

Materials

QIAprep Spin Miniprep Kit (Qiagen 27106)

E. coli culture


Protocol

This protocol is adapted/condensed from the Qiagen instructions. The Qiagen site ([1]) offers several helpful tips that are useful for first-time users and for troubleshooting.

1. Grow 1-5 mL of E. coli for 12-16 h at 37ºC.

2. Pellet culture and remove supernatant (3 min. at >6800g is sufficient).

3. Add 250 μL of resuspension Buffer P1 to pelleted cells in microcentrifuge tube and resuspend cells.

4. Add 250 μL of lysis Buffer P2 to tube. Invert several times until solution is viscous and homogeneously blue (or slightly clear if no LyseBlue was added).

5. Add 350 μL of neutralizing Buffer N3 to tube. Invert several times until suspension is clear and clumps form.

6. Centrifuge at 15,000-18,000g for 10 min.

7. Add supernatant to a QIAprep column in a collection tube by pouring or pipetting.

8. Centrifuge the column at 15,000-18,000g for 30-60 s and discard flowthrough.

9. Add 750 μL of wash Buffer PE to column,

10. Centrifuge the column at 15,000-18,000g for 30-60 s and discard flowthrough.

11. Centrifuge the column at 15,000-18,000g for 60 s and discard flowthrough (to eliminate residual ethanol).

12. Place QIAprep column in 1.5 mL tube.

13. Add 50 μL of elution Buffer EB to the center of the column. Let stand for 60 s.

14. Centrifuge at 15,000-18,000g for 60 s.

15. Store DNA collected in vial at -20˚C.


Note: If the strain has high carbohydrate content or high nuclease activity (such as an endA+ strain) wash with 0.5 μL of Buffer PB before PE wash.

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