Skip to content

Latest commit

 

History

History
33 lines (20 loc) · 635 Bytes

backbone-prep.markdown

File metadata and controls

33 lines (20 loc) · 635 Bytes
Raw

pET29 backbone prep

  1. Grow up a 200 mL culture of E coli harboring pET29

  2. Maxiprep the resultant culture to obtain 2 x 500 uL aliquots of purifed plsmid at ~ 200 ng/uL

  3. Perform the following digest

75 ng / uL * 75 uL => 5,625 ng 200 ng / uL * 500 uL => 100,000 ng

Reaction conditions: recommended

5-10 units each enzyme + 1 ug DNA in 50 uL

(enzyme volume not to exceed 10% of total reaction volume)

NdeI and XhoI for 1 hour at 37

So for 50 ug of pET29

250-500 units enzyme + 50 ug DNA in 2500 uL

AKA

12.5-25 uL NdeI + 12.5-25 uL XhoI + 250 uL CutSmart + 500 uL DNA + 1700-1725 uL water