lenght of my inserts vary from 450 to 1.5 kb. all amplification primers have CACC at 5' of forward primer. after topo reaction(followed as instructed in manual) got colonies. when i did colony PCR for almost every type of insert, every colony is positive.
when i isolate plasmid and give it for sequencing, surprisingly no insert at all !. sequencing results shows intact attL1 but modified (rather should say deleted) attL2. same happened for about 30 types of pENTR D TOPO reactions (i.e 30 different insertions).
your suggestions are valuable.
Hi
as I know the primer at the annealing temperature require only 4 nt to anneal. So if you check the annealing step in your PCR reaction ( you might need to increase it ), the sequence of the plasmids that might have sequence matches your CACC.
did you conducted any positive control ( an insert that is usually worked with your vector at the same PCR).
I do not know what kind of reporter that you use for you plasmid, but if you make sure of your reporter gene in the plasmid that might help.
Wish you luck
hi mohammed,
thanks for reply. after amplification of my gene (one of the e.g., is H2B of arabidopsis) gel purified and used for cloning. any of my clones does't have any reporter gene.
the only step i did't take is using template provided by them as positive control (to save topo vector) but at least some clones should have worked.
this time i try with positive control and see what happens!
but it is still big puzzle to me why attL2 is just gone out of plasmid.
I suspect your colony PCR. Are you keeping a negative control?
Please do a restriction digestion to release the insert as conformation before going for sequencing.
Look at the discussion at the following link
https://www.researchgate.net/post/What_is_the_best_way_to_select_and_confirm_clones_prior_to_sequencing/1
Hi,
I agree with Panduranga.
Have you digested your product?
During Colony PCR keep a nevetive control.
Check the pENTR kit with positive control.
You can also increase the Reaction time up to 30 minutes.
@paramita ghosh
thanks for answer. i keep reaction for overnight incubation.
hi,
I usually keep the reaction for half an hour. few months back, we faced a similar problem with pENTR kit, but even the positive control did not work. We consulted with the company and got replacement. However next kit that we have received is working fine. I have been working with gateway vectors for last 1.5 years. 450 to 1.5kb is not a very big inset, so it should not give problem. I don't know your reaction conditions that you might have standerized, we usually use 2:1 molar ratio of PCR product: TOPO vector. PCR product between 30-40 ng/µl.
And sometimes you get false colonies too after antibiotic selection. the morphology of those colonies are little different if you observe closely.
Here are some tips to get colonies:
. high concentration of pcr products (up to 60 ng/uL)
. gel purification
. 15-30 min reaction
. do not mix by pipetting up and down the bacteria
I used pCR8 GW TA for sequencing and worked fine
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