cloning protocol - (Nov/16/2009 )
Hi, can anybody tell me were I can get a protocol to clone pcr products with puc19 from fermentas_
First of all, you have to specify if you are using a proofreading polymerase for your PCR, like Pwo for example, or a polymerase that doesn't correct mistakes, like Taq. This is important because Taq leaves A tails at the end of the PCR products that unable you to clone into pUC19, unless you treat the product with another enzyme to remove A tails first and make blunt ends.
So I assume that you are using a proofreding polymerase for your PCR. In that case, you can follow these instructions:
- Cut pUC19 with SmaI or another restriction enzyme that leaves blunt ends. Purify the linearized vector. Quantify the amount of DNA to set up the ligation ratio that you want (3:1 -insert:vector, for example. People generally use a higher proportion of insert)
- Quantify your PCR product after purification, and add the amount of product that you want for the ligation reaction.
If the proportion of insert is too high, you will have more than one insert cloned into your vector, so it may be useful to try different ratios.
I make ligations in a final volume of 15 µl, at 16 ºC, O/N.
Transform DH5a E. coli cells, and plate the transformation on LB+Ampicilin plates. Remember that before plating the bacteria, you have to put X-gal+ IPTG, because otherwise you will not be able to select the positive colonies.
In the morning you will see plenty of colonies. The white ones should be the good ones!
To avoid having so many blue colonies, you can also phosphatase the vector (pUC19) after digestion with SmaI. But if you do this, take care because PCR products do not usually have phosphates at the end of the molecules, so without kinasation of the primers before the PCR reaction, ligation with the vector will not take place.
So if you phosphatase the vector, make sure that you have also kinasated the primers!
This is what I do for cloning PCR products into pUC19. It is easy and cheaper than using kits. I hope it helps you.
(Sorry for my English mistakes!)