I have been trying to clone a coding region of my gene in pdonr using gateway method. I successfully cloned CDS but when I sequenced the colony the region from the middle of about 100 BP was missing. What could be the possibly reason of that? Note: It doesn't have any other transcript. What should I do to overcome the issue?
Similarly, for promoter and coding region together, get colonies on zeocin resistant plates but I don't get positive colony PCR with specific primers. What should I do?
The gene may have been deleted during the cloning process or damaged during the sequencing process.
Clone and sequence again and you will know if the gene exists or not.
Determine gene is properly ligated into the plasmid and the sequencing machine is in order.
The primers may not be specific enough, the reaction not long enough, or hot enough. The DNA could be damaged.
Try a different PCR machine if possible.
Hi,
I would suggest to get it cloned from genscript. They will synthesize, clone and seq your whole plasmid with nominal cost. Comparing time, effort and money, getting is done by company is better option. Need help with that, get back I can put you through the right channel.
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