Want to link mCherry gene in pHB vector but every time not getting results.., Restriction Enzyme digestion was successful for both vector as well as insert gene (confirmed on gel and picture clearly showed slow movement of digested plasmid)., after ligation and transformation reaction all colonies were neagtive...is there self ligation or what can be the issue ..?? or issue is with the pHB vector.....
How are you testing your colonies? If you are doing colony PCR, I'd switch to an overnight broth culture, plasmid mini prep, digest to linearize and then PCR for the insert.
How many colonies have you tested? Sometimes it just takes screening a lot of colonies to find what you need. If you've looked at less than 30, keep looking.
Dear Shahnas,
ligation might be a problem; you would change your ligase and ratio you use.
Generally I used to screen 40 colonies and if not get any positive I test 40 more with colony pcr. I would suggest you using heating method for colony PCR. It works better.
However, before all that you might see Mcherry colonies pink on selective plate before you even test them in colony pcr.
good luck...
@Ali Akgul...you mean if mCherry is expressing it should give red color ? ok actually for ligation I used 2 kits.... from takara and NEB ..,, but still problem....for colony PCR I am checking around 30 clone but as you people suggested I will increase number now ....Thanks
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