I have been trying to clone 1.5 kb PCR fragments with A-overhangs (High processivity, low fidelity taq polymerase used). But each time I am not able to get the clones.
In the first time experiment, I carried out the ligation at 4 degree celcius overnight in a thermal cycler, and then carried out the heat shock at 42 degree celcius in a thermal cycler for 1 minute. The reaction was carried out in 0.2 ml PCR tubes with 50 μl of JM109 competent cells each. Ligation reactions were set up as 1:1, 1:2, 1:3 vector to insert ratio along with two positive control using pGEM-T vector + control DNA provided in the cloning kit and with uncut circular vector DNA provided with the JM109 competent cells kit. 50 μl transformed bacteria were added with 950 μl LB broth, incubated at 37 degree celcius for 1.5 hrs and then 50 μl plated in x-gal, IPTG LB plates with and without ampicillin.
Result: Colonies developed only in the LB plates without ampicillin but no colonies in LB with ampicillin.
In the second experiment, I carried out the entire same protocol as depicted in the manufacturer's manual making the following changes- the heat shock was carried out in 1.5 ml eppendorf tubes against 0.2 ml PCR tubes and in a water bath set at 42 degree for 2 minutes as against thermal cycler set at 42 degree for 1 minute as done previously. This time the 50 μl transformed cells were incubated in 950 μl SOC media as against LB broth for 1.5 hrs at 37 degree shaker.
Result: Colonies developed only in the LB plates without ampicillin but no colonies in LB with ampicillin
Try troubleshooting your transformation protocol before worrying about the ligation step. One to two minutes seems excessively long- typical protocols are 45-50 seconds for JM109 (I recommend 45sec). Use pUC19 or another positive control plasmid to make sure your competent cells and transformation protocol are working with the expected CFU frequency. I suspect that either your competent cells are no good, or your heat shock protocol isn't right (for example, too long a heat shock).
Do you purify your PCR product over an agarose gel before ligation? this sometimes helps. Additionally, I use 9:1 Taq:Pfu for my PCR which work quite as you have both, speed and fidelity. We use DH5alpha chemo-competent cells and the transformation with pGEM-T ligations works without problems. In my experience it does not really matter the ligation conditions or heat-shock time.
Hi Rajal,
Did you verify by agarose gel if the vector or the insert are degraded before ligation?
@Isabel: No i dint not cross check my ligation product prior to transformation.
Hi Rajal,
sometimes the vector and the insert can be degraded during the purification of the insert or the storage of one of them. For this reason i always check both in a gel prior to ligation.
Hello Raja,
My name is Robert Deyes and I work with Promega's Technical Support Services group. I am writing to find out whether you have been successful in obtaining pGEMT clones.
A point that I would like to bring up is that I have occasionally found inserts to be toxic to E.coli (ie bacteria just do not grow after transformation). One way around this is to do your overnight growth at a lower temperature (eg 30 degrees). You will get much smaller colonies that way (pin prick colonies that can only be seen by holding the plate up to the light). But they will be positive colonies nevertheless.
Another option might be to transform into a bacterial strain that is specifically designed for difficult-to-transform inserts/plasmids. Agilent Technologies sells SURE cells for this purpose (see http://www.genomics.agilent.com/en/Competent-Cells-Difficult-Cloning/Unstable-Clones/?cid=AG-PT-118&tabId=AG-PR-1218&Nty=1&Ntx=mode+matchall&Ntk=BasicSearch&N=4294967292+4294967234+4294967294+4294967244&type=baseSearch&No=0&Ntt=SURE).
You would do your ligation the same way, then transform through heat shock into E.coli instead of JM109.
Please let me know how things go.
Many thanks,
Robert
Robert Deyes
Technical Services Scientist
Promega Corporation
Tel 1 800 356 9526 Ext 1366 (USA)
Tel 1 608 274 4330 Ext 1366 (Long Distance, Toll Paying)
http://www.promega.com
Hello Robert,
Thanks for the input. I have succesfully standardized it, I changed the JM109 cells to DH5alpha cells. and did a bit of manipulations as suggested by others.
Thanks a lot.
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