Ligation of purified PCR product to Topo plasmid (with T7 promoter) and Transfor - (Jul/06/2007 )

Hi everyone,

I am trying to ligate my PCR product to a Topo plasmid with T7 promoter). Both the plasmid and PCR product have been double digested with Nde1 and BamH1. The ligation protocal is as follows :-

10 microlitres of T4 ligase buffer
2 microlitres of T4 ligase
6 microlitres of digested PCR product
2 microlitres of vector.

After I have set up this ligation I leave it to stand for half hour at room temperature and then transform. Transformation protocal :-

5 microlitres of ligation to 200 microlitres of competent cells
(My competent cells are always thawed out on ice)
leave on ice for 30 mins
heatshock at 42 degrees for 1 min
chill on ice for 2 mins
add 795 microlitres of LB
incubate for 1 hour at 37 degrees
plate out 200 microlitres onto amp plate and incubate overnight at 37 degrees.

I have tried this transformation 4 times now and I have also set up controls with uncut plasmid and competent cells.

I have found that I always get colonies on my control plate but noen on my experimental plates. I have tried using a different T4 ligase and ligase buffer but this has made no difference.

I have also tried to cut the plasmid with just Nde1 and tried to religate and transform however this has failed as well.

Any ideas on how to make this work?

Any help would be much appreciated

Thanks

Worried 2

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Hi there, firstly, I need to confirm something. Is your 'TOPO vector', 'TOPO' plasmid one that your lab has made and has been circularised. And has said 'TOPO' been digested by both NdeI and BamHI?

When one mentions TOPO vector, one imagines TA cloning with the added help of topoisomerase. TA cloning is used to clone PCR products that are A tailed. Said PCR products are not digested by restriction enzymes.




The above ligation reaction, again I must ask, has the ligase buffer been dilute to 2X? Ligase buffer often come in as 10x more concentrated. So a 20ul total volume reaction, needs only 2ul of 10x ligase buffer. Secondly, 2ul T4 ligase is a little too much. More is not better here. I would suggest that you trim down the ligase to 0.5ul unless again the T4 ligase has been diluted.




I would ligate the reaction overnight, unless you are using quick ligase. Which in turn means that your have to remove the PEG that comes with the ligase buffer, as PEG is inhibitor towards ligation efficiency.




I am no expert with chemical transformation. But I would switch to SOC for cell recovery. And trim the recovery time to 30 min and no more then 45mins. 1hr is a bit on the long side. Furthermore I would plate out the remaining 800ul on a second plate, just incase effiency is low. And there is no need to thaw for 30mins! The cells thaw rapidly. Hmm is 200ul of cells a bit much.... need comments from people who do chemical transformation regularly. Are these homemade or company made competent cells?



Probably indicate a problem with the ligation reaction. Also another thing the PCR product, how many bp were used to skirt the NdeI restriction site. NdeI requires a larger then usual skirt for the NdeI restriction enzyme to cut efficiently. 8bp is about minimal. Look up NEB's technical guide on cutting close to end with restriction endonucleases.

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Hi ,

The TOPO vector is the vector that my lab has recirculised. The competent cells are home made they are not from a company.

Thank you for your reply.

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