I am performing experiment on E.coli DH5 alpha transformation using PCR 2.1 TA vector I use fresh PCR product and efficient competent cells but I am not successful in achieving positive transformation.
a. I am using 10 µl ligation mixture of PCR product : 4 µl, PCR 2.1 TA vector 2µl, Ligase 1µl, Buffer 2µl and water 1 µl. Kept at 4˳C overnight and perform transformation on the next day.
Can anyone help me to optimize it or to indicate the problem.
b. How to convert 30 ng of PCR product in microlitres?
For Part A, you're using the wrong temperature. https://www.thermofisher.com/us/en/home/references/protocols/cloning/ligation-protocol/cloning-of-a-tailed-pcr-fragments-using-conventional-ligase-method.html
Also, make sure your DNA polymerase leaves an A overhang on the product.
For part b, you need to know the concentration of your PCR reaction (how many ng of DNA are in each microliter). Use a nano drop or do an "old school" estimate on an agarose gel compared to a DNA ladder with known concentrations of each band.
C1V1 = C2V2
Hi, I agree with Katie A Burnette , but would add some information.
regarding part b.
As I understand:
- You have to use 4 ul of PCR product
- You decide to use 30 ng of amplicon (in 4 ul)
Precision is good idea, but I am not shure that it could solve your problem, but...
Please try to measure amplicon (dsDNA) concentration using fluorimetry (Promega Quantus, Thermofisher Qubit etc.). In PCR tube you can find many compounds including primers and dimers of primers, dNTPs and etc. All this can affect the results of measurement of concentration. Fluorimetry is better than spectrophotometry and agarose gel even with gel-doc automated systems (I think).
Usually you can find a lot od DNA after PCR. And it is very probably that you have to dilute your amplicon to receive only 4 ul. Be carefull.
30 ng/ 4 ul = 7,5 ng / ul of ds DNA. It is very low concentration. You have to dilute your PCR product.
I hope it helps.
How long is your PCR product?
Basically, the molar ratio (PCR product:vector = 3~5:1) is one of the standards in the ligation step, following the manufacturer's instructions for the ligase.
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