I have cloned my gene of interest in pSpeedET using NgoMIV and Xba1 restriction enzyme. Gene was isolated via PCR using gene specific primers with restriction site over hangs at 5' ends. After ligation and transformation I got many colonies on selective agar (Kanamycin) from which i screened 5 colonies via colony PCR using a) gene specific primers, b) T7 forward and gene specific reverse primer but i didn't get my specific PCR product. Protocol i followed for colony PCR is:
1) diluting colony in nuclease free water(50uL)
2) heat denature at 95 C for 10 min.
3) spin down at 10000 rpm and harvest supernatant as template for PCR.
NOTE: pSpeedET contains ccdB(Toxin) cassette due to which unligated plasmid (intact ccdB gene) cannot result as transformant i-e for transformants they must have interrupted ccdB cassette by insert.
Kindly suggest me the possible faults i might have gone through.
Thanks in advance.
Seems fine, actually. Did you try to amplify a large fragment in the colony PCR? I've had myself problems with fragments >500 bp in that setting. In such a case, you may need to order an extra reverse primer inside the insert, such that the PCR product with this primer + T7 forward
Shehzaib, when I do colony PCR, I resuspend the colony in 50ul H2O as you do, but take 1ul of the suspension straight into my 20-25ul PCR mix (I add 1ml LB+antibiotic to the rest and keep at 37C; I grow the ones that turn positive). I have never had any problem. You are right about your assumption on ccdB gene function. But what you have not told us is how you prepared the vector digest. Many people go for a short-cut: digest the vector, deproteinize, and use in the ligation reaction. The trouble there is, an inadequate digestion will leave a good number of supercoiled vector. As you already might know, supercoiled plasmids have far greater transformation ability than the nicked/ligated ones, and they will easily outweigh your constructs. So, I suggest you always gel-purify the linear digest. Second, you have not told us if you get amplification for an internal, vector-alone region. Make a pair of oligos to amplify about 150-300bp from the vector backbone and make sure that these primers do not interfere with the pair you use to detect the ligants. Keep the Tm for all four primers close enough so that they all will amplify in the same reaction. Throw in all four primers in each reaction; this will give you an internal control (your positives clones will have two bands while the negative ones will have a single band; but make sure that the length of the bands are about/at least 100bp apart); run a high-percent gel so that you can see the separate bands quickly. Besides, miniprep takes just a couple hours max and a diagnostic digestion another 30min. So start with, say, 10 colonies and with isolated plasmids.
Shehzaib, You have not mentioned what was the size of your insert which you have ligated. I suggest that you can use your gene specific primers as well as T7 forward primer and the primer specific to the sequence lying at the 3' end of vector (i.e. T3 or SP6, etc. )?
Next time, don't bother to resuspend your colony before adding to your PCR mix. Just take a 20-µl pipette tip and use it as a toothpick to pick a little bit of your colony, patch on a plate (to recover your positive clones) and resuspend the rest of what is left on the tip directly into 10 µl of your PCR mix. Don't overload. Perform the PCR cycling (25-30 cycles) and load 4 µl on an agarose gel. I use pipette tips because they are sharper than toothpicks, disposable in large quantities and thus unlikely to be contaminated with any kind of unwanted DNA
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