Ligation - Biology Forum

last updated by

Navin 18 years, 5 months ago

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- June 11, 2006 at 3:11 am #5010
  jamesgoat
  Participant
  I tried to transform JM 109 with pGEM-T vector (w/ inserts). But when I plated them on LB/Amp/X-Gal/IPTG, there were only 3-4 colonies of each blue and clear colonies coming out. Wondering what has happened. Will it be the ligation problem or JM109? I did positive control and it comes out ok. Does anyone experience this? Thanks in advance.
- June 15, 2006 at 11:16 am #50098
  oppox
  Participant
  It doesnt matter if u get only one colony as long as its a positve one. I got about 15 about half was correct.
- June 15, 2006 at 12:03 pm #50102
  Navin
  Participant
  I did an experiment before involving restriction digestion, ligation and transformation and I had expected many colonies to form but only 1 did. Then my mentor told me (after much thinking) that this most probably occured because the restriction enzyme I used was very inefficient and may not have cut the vector – so only 1 colony formed. So now, I am increasing the amount of the restriction enzyme and I hope that I get many colonies once I transform them.
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