I am doing almost exactly the same thing as Mr./Ms. dorightman did. I hope you won't mind my copying the description:
PCRing my gene (1.5kb) out from a cDNA library template to create different restriction enzyme sites at the ends. I gel purify the PCR product using a kit from Qiagen and elute with 10mM TrisCl, pH8.0. I get a good recovery....i check this by running it out on a gel. from this estimated conc. i digest with NdeI and EcoRI and gel purify and test conc. the same as above. from this i set up a ligation in a vector:insert ratio of 3:1, 1:1, and 1:3 with ligase from Fisher. i transform all 10ul of ligation reaction into 100ul of competent cells and plate 100ul of the transformed cells, but still get no colonies.
I actually check my PCR product by doing PCR again with exactly the same primer pair. However, I didn't get any PCR amplification again. Could some body help me with this? Am I supposed to get a lot of amplification from it?!
PCR product for ligation
1 reply to this topic
Posted 01 October 2004 - 05:17 PM
I actually check my PCR product by doing PCR again with exactly the same primer pair. However, I didn't get any PCR amplification again.
Probably you have used too much template?
You have to do a positive and negative control transformation before others can shoot the trouble for you.