screening of positive clones - colony PCR /Plasmid isolation and Restriction digestion (Feb/12/2010 )

Hi all,

I have done cDNA library construction. and now I am stuck up with screening the positive colonies. I did colony pcr with M13 primers, it is giving some amplification of different insert sizes. thats good but when I isolate plasmid by qiagen miniprep kit nothing is found on gel. hope some one resolves this problem.

ThanX

Nothing, as in no DNA, or nothing as in no insert in the vector?

Hi,

Is the size of the colony PCR is what you targeted? If the answer is 'yes' perhaps you could troubleshoot in your plasmid extraction step. Otherwise, I would suggest that you repeat the cloning step.


Cheers,
appleyun

Hi,

Actually the colony pcr Iam doing is for cDNA library constructed (for which we do not know the pcr product size to be amplified). I am using M13 primers to amplify the pcr product . Now, Iam getting the pcr bands but no plasmid observed after plasmid isolation.please any one help me.

thanX in advance

If you isolated plasmid from Yeast cells, it's big chance you can't see the plasmid from the gel, but you can send it to sequencing.

novagen on Feb 28 2010, 07:51 PM said:

Hi,

I'm having quite the same problem, when I sequenced my positive clones from cDNA library, it is hardly hit the targeted organism. What I did was from the cDNA library (lambda TriplEx2 cDNA library) I do immunoscreening to screen for positive clones using panel of serum samples against parasitic disease. When the potential clones were obtained, I proceeded with in vivo excision to excise and convert the phage into plasmid form before it was subjected to plasmid extraction prior to sequencing. The sequencing results was so dissapointing, I expect to get the right sequence (at least 80% of the clones hit the target) since the clones was quite specific when tested with serum samples. I'm not sure which step I've done it wrong, whether the in vivo excision or plasmid extraction that caused the sequence cannot be read.

Hope someone can help me

Thanks