I'm doing the gateway cloning from PCR product. I first ampliflied my interest gene with the attB1/2 and then performed the BP clonase reaction to construct the pDONR211 vector with my inserted gene. The problem was I got the transformant on the LB+Kan agar, that's mean the ccdB gene at the insert site has already gone but when check with M13 primer set, there is no insert. How can I solve this problem. Thank you very much.
Hi,
Did you check if your ccdB gene is mutated? There are higher than normal mutation rates on 5' donor vectors and sometimes ccdB gene with GATEWAY system. If you haven't done already, I'd suggest transform pDONR221 vector alone and check on Kanamycin plate. Also, inoculate non-transformed cells into LB-Kana to check if your kanamycin is still OK. One last thing you can do to screen as many colonies as possible. If 99 of your colonies doesn't contain insert but one does' you don't need to worry about it any more and proceed with it.
Hope I could be any help.
Hi,
Is there nothing between both recombination sites? Not even a very small primer-derived sequence instead of your desired insert?
You could try do identify colonies with larger inserts via colony-PCR using the sequencing primers. The resulting PCR products will show you the approximate insert size. However, it could be easier to improve the PCR for the insert generation and the BP reaction. The BP reaction was inefficient if you observe lots of "empty" plasmids. Have you purified your PCR product prior to the BP reaction? This could help to remove small fragments.
The ccdB gene is probably not the issue, because you observed plasmids without an insert. Therefore, only cells with "empty" plasmids survived your selection. The ccdB gene is responsible for killing all the others. A mutation in the ccdB gene would cause many colonies with the original plasmid.
Good luck with your cloning!
Thanks everyone. OK I finally got desire clone with my inserted gene but I have another problem. After done DNA sequencing using M13 primer, first 130 bp of my gene is not belong to my interesting gene. Let's say my gene is 2064 bp in lenght but only 131 - 2064 bp is my genes -1-60 bp is some gateway vector sequences, 61-90 bp is attB1 same as I the primer I used and from 91-130 is unknown. I don't know where the problem come from??
Could you please provide your gene of interest, the primer sequences and the sequencing results?
Thank you very much. I found out that there is some strange sequences attached with the primer (I didn't design) so I designed the new and successfully construct a pDONR211 vector.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Methods
- Cloning