wrong sequencing result?! - (Sep/15/2006 )
Hi, thank you for your advice. Yeah, the 800bp is from another plasmid constructed by a lab member.
However, if I recovered the wrong fragment, why the following enzyme digestion worked well?
Today I just went on a PCR test of the new constructed plasmid, BUT I did not get the desired band around 800bp. The same sample for enzyme digestion works well and it seems that the two enzymes can cut it well. So during which process did the problem begin to turn up?
You've got an 800-bp fragment whose sequence doesn't match what you expected, but does produce the right restriction pattern.
I'd suspect strongly that you cloned the wrong fragment.
Why don't you PCR amplify the fragment you want using your labmate's plasmid as template, and clone the PCR product?
I'll suggest that when analysing your gene sequence analyse it from both ends first in a forward direction which I believe is giving you the incorrect sequence and then in the reverse direction maybe you'll get it right.
Keep us updated on the developments.
Thanks so lot!
I have already aligned in the reversed direction and it did not match. So my labmate suggested that it may be plasmid contamination after enzyme digestion. Since the constructed plasmid can be digested, but after that seems that it have changed to another plasmid.
Now I am just going to re-start from PCR amplification. Try to be very careful this time to avoid any potential contamination.