Unable to ligate two PCR products - (Aug/20/2007 )
I have been trying to ligate two PCR products (1.7 kb and 0.9 kb) with no luck. The fragments were amplified with Phusion enzyme. They both showed up nicely on the gel. Each PCR product has a BamHI restriction site at one end (with two extra nucleotides at the very end). I digested my PCR products at 37C for 2 h (100 units of enzyme in 100 ul of reaction mixture), cleaned them up, and did the ligation (10 units of ligase in 20 ul of reaction mixture) at room temperature for another 2 h. When I ran the ligation mixture on the gel, there was no extra band showing up.
I checked DNA concentration using Nanodrop every time after cleaning so I knew the DNA molecules were there. I made several attempts by increasing the incubation time (either digestion or ligation or both) to 16 h, and it did not help. Indeed the best result I got was when I shortened the digestion and ligation step to only 1 h, which gave me a very faint band of the right size (2.6 kb) on the gel.
Could anybody help me out? It should be a simple step but I have been stucked for 2 weeks. Any suggestion is welcome.
You don't say how many micrograms of insert DNA you are digesting with BamH I. The 2 basepair clamp on the end works better if it is composed of Gs and Cs, as AT ends tend to breathe.
1 microgram of a 1700 bp PCR fragment = 0.905 pmoles and has 5.45 e 11 BamH I sites.
1 microgram of a 900 bp PCR fragment = 1.709 pmoles and has 1.03 e 12 BamH I sites.
I have routinely digested 14.4 pmoles of PCR fragment in a 150 uL reaction with 80 Units of BamH I for 3.25 hours. This means that you can digest about 3.4 e 11 molecules with 10 Units of enzyme per hour.
I use 14.4 pmoles because 25 micrograms of pUC = 14.4 pmoles and has 8.8 e 12 BamH I sites. The pUC digestion yields enough vector for many ligation reactions. The vector is simultaneously treated with SAP to prevent ligation of linearized (single-cut) vector. 14.4 pmols pUC9/25 µg plasmid DNA x 2 ends = 28.9 pmoles of 5’ ends x 0.1 unit SAP/pmol = 2.9 units SAP/25 µg linearized pUC9.
If you are using 10 NEB Units of ligase, you may get better results with an overnight ligation. If you are using Weiss Units, the 2 hour ligation should be sufficient. 66.7 NEB Units = 1 Weiss Unit.
Just checking. You did purify your PCR product *before* the digestion, right?
Another question I must inject, how is your dephosphorylation of your vector going? What conditions have you used? How much DNA was dephosphorylated and for how long.
May be it would be better to clone into pGEMT, pTOPO or similar first for a better digestion.