PCR cloning problem. Pls help...very urgent! - PCR cloning problem. Pls help...very urgent!!!!!!! (Oct/18/2008 )

I have amplified my pcr product using Biotools taq DNA polymerase using 2mM MgCl2 and the final extension step was at 72deg for 10mins to add the A overhangs. I then ran the pcr product on 1% agarose gel and had a strong band of desired size (1450bp). I cut out the band and gel purified it using Geneaid gel extraction kit. For my vector, I digested puc19-TA (2.6kb) with xcmI and ran the digested products on agarose gel and extracted it. I then measured the DNA concentration of the pcr product and vector using UV spectrometry and set up ligations reactions encompassing 1:2, 1:3, 1:5 molar ratios. After transformation and plating on LB amp plates, I found that the control plate had many colonies (about 50) and test plate had slightly more (about 70). When I checked 6 colonies on the test plate, all only contained the vector. I repeated the above assay many times differing ligation temperatures, different molar ratios but each time I get strange number of colonies. Sometimes my control has no colonies and my test has 10 and all the colonies only contained vector.

Then I read a paper by Qiagen which mentioned that if the 5' terminal base of the primers is an A or G, the terminal transferase activity of the polymerase will be high. The 5'end of my forward primer was a C and so I used another primer which has an A at the 5'end. But again, it did not give me positive clones.
I decided to forgo TA cloning and tried a different strategy. This time after pcr and gel elution (eluted in 15ul), I cut 4ul of my pcr product with xhoI and bamHI and ran products on gel. I cut out the desired band (500bp) and ligated with another suitable vector cut with the same enzymes. Yet again it only gave me clones with only the vector present. I thought it might be because I lost much of my insert molecules when gel eluting. Thus I tried another way. This time after pcr I ran a gel and eluted out the pcr product in 15ul. Then I use 10ul to digest with the same enzymes. After digestion, I did not run a gel. I used the digestion mixture directly for ligation but it did not give me positive clones as well. I also realised the digestion of my pcr products is never 100% complete as I can see a very very faint band of undigested DNA on the gel (which I don't think would affect the ligation since I cut out only the digested product from the gel).

Then a few days ago, I realised that for pcr I actually accidentally used 2mM of primers instead of the standard 0.2mM. So, I did pcr again using 0.2mM primers and tried to clone using the restriction digest method but it still did not work. I have yet to try TA cloning using pcr products amplified with 0.2mM primers. To add on to my problems, I read in the protocol by Fermentas that having an A at the 5'end of the primer leads to the worst terminal transferase activity of polymerase. This contradicts with that paper by Qiagen which I read earlier.

My fellow lab mate managed to successfully clone her pcr product using the restriction digest method. So, I am confused now why both TA cloning and restriction digest method is not working. Any idea what could be wrong? Why isnt the ligation working and why are there so many colonies on my control plate even when the ends are not compatible? I only have about a month more to get my clone!!!!!!!!!!

Note:
1. My lab does not do phenol extraction for pcr products coz gel extraction itself removes contaminants and it works for my lab mates.
2. Ligation is usually done overnight at 4deg.
3. We do not regenerate in LB media prior to plating coz all the while, colonies are produced even without regeneration.

check for ligase activity by running ligation with HindIII digested lambda DNA ladder ( serve as a positive control)

it could be a few things ..

ur ligase is bad
or your DNA quality is bad. ( what's the A230 of ur DNA ? ratio wise with A260:A230 is how much?)

The ligase is working fine because my other cohesive end ligations are working well with the same ligase. The vector and insert molecules also seem ok in terms of quality as A260/A280 is bet 1.5 -1.9.