5 replies to this topic

[Enzy]

Hi all,
i am having a problem in ligation of blunt ends. I have used BamHI and SmaI for digastion.
how to solve this problem
please suggest me some ideas!
thanks in advance!

[rkay447]

Eggads my friend...you are going to have to give us more information so that we can help figure out where things went wrong!!  So, let's start at the beginning.  Are you trying to take a pcr product and ligate it into a vector or are you digesting an insert out of one vector and moving it to another vector?  Are you doing a sequential digest?  SmaI is a tricky enzyme that is deactivated very quickly at 37degree, which is what you need for BamHI so you should be doing sequential.  How are you purifying your insert and vector?  What enzyme are you using for ligation (T4 or quick) and under what conditions have you tried?  What are your results?  No colonies or lots of empty vector colonies?  Are you sure your insert and vector are at the correct concentrations?  Have you run the two out on a gel to see them and know that they are clean, at the correct concentration?  Please take us through what you have done and what the results were so we can suggest where you can make changes.  Also, please include the sizes of your insert and vector and the amount of each you put into the ligation reaction.

Edited by rkay447, 10 June 2010 - 04:04 PM.

[Enzy]

Dear Friends,
Thanks for the reply.
I am using 9.5kb vector with BamHI and SmaI separated by 8bp, as a vector backbone. I am cutting this vector sequentially with these two enzymes. After each digestion I am purifying the digestion sample using Qiagen PCR purification kit. Since the release after digestion is only 8bp I could not see it on the gel. The insert is 3kb long and is digested out from vector using SmaI and BamHI(unique sites) enzymes at 28 degree O/N. The insert was gel purified and it was looking fine when I checked in the gel with marker. This insert was ligated to vector in 1:3 vector:insert ratio using T4 DNA ligase at 16 and 4 degree O/N. The ligation mix was purified,checked in the gel ( it was looking like ladder) and transformed to E-coli DH10B competent cell. I have screened all 25 colonies and all of them have only vector backbone. Please tell what to do the solve this problem. I have to use this strategy for cloning this insert. How to make ligation successful or is there any way to remove the self ligation of vector( I have tried dephosphorylation)???

Thanks in advance.

Edited by Enzy, 13 June 2010 - 04:17 AM.

[pDNA]

your problem seems not to be religated vector but uncut vector instead!

Try gel purifying your vector backbone instead of PCR purification ...normally this severly reduces background.

Regards,
p

[Enzy]

pDNA, on Jun 13 2010, 02:39 PM, said:

your problem seems not to be religated vector but uncut vector instead!

Try gel purifying your vector backbone instead of PCR purification ...normally this severly reduces background.

Regards,
p

Thanks for the reply.

[dpo]

pDNA, on Jun 13 2010, 02:39 PM, said:

your problem seems not to be religated vector but uncut vector instead!

Try gel purifying your vector backbone instead of PCR purification ...normally this severly reduces background.

Regards,
p

I second that. Trace amounts of your orginal plasmids can cause a lot of problems.

Additionally, do you have access to XmaI? This has the same recognition site as SmaI, but leaves a sticky end, always easier to ligate than blunt ends.