Cloning for Bisulfite sequencing - TOPO TA no results...... pGEM???? (Mar/27/2007 )

Hi All
I have to bisulfite sequence 3 different genes in 8 samples.
So far.... I have successfully cloned and sequenced 1 gene that was 700bp using the TOPO TA kit. This was my 1st attempt and everything went fantastic (all within 3 weeks).

And now its close to 2 months and I haven't been able to get any positive clones for the other 2 genes - both around 250bp! I am aware that smaller products SHOULD clone much more easily than larger ones!!! I have run ~450 colony PCRs in all - got 50 positive, could revive 35 - and 15 showed up as positive by restriction digest! Then I just did crude mini preps 137 and got 7 positive!!! (Just not willing to give up without getting my clones - so insanely hanging on to useless colonies )
I am receiving my second replacement kit from them............ in one attempt we found the competent cells were not really competent. But none of the techs could explain why I had so many +ve without my insert.

Now I am trying the pGEM-T easy vector system from promega along with the TOPO kit that i will be receiving!

Our sequencing facility uses the ABI PRISM 3130XL Genetic Analyzers - and they suggest -
"Host strains that generally give reliable data include DH5alpha, DH10B, DH1, C600, XL1-Blue, XL10-Gold, TOP-10 and NM294."
"Strains such as JM101, JM83, HB101, TB1 and TG1 are NOT recommended as they can release large amount of inhibitory factors during lysis that can lead to poor quality DNA that will require extra purification steps."

I was wondering which cells work well with pGEM system. The tech could not tell me if the JM109 cells would work any differently from the JM101 cells.
Anyone with success using the pGEM system what cells do u use?

Out of curiosity how many of you calculate a 3:1 insert:vector ratio even with the TOPO TA kits?

Thanks - a lot of the previous queries on colony PCR etc were helpful in a number of ways
But I need more help

hi learning,

we use pGEMT easy kits here with DH5-alpha cells and they work rather well. However we do see a number of false positives and primer dimer sometimes, so it's very crucial to clean your PCR amplicon up properly, I tend to gel extract and use the SV gel extraction kit.

The 3:1 ratio is to ensure an excess of your amplicon to your plasmid, I go way above this to ensure that every plasmid has been ligated to an amplicon.

Also, I tend to use much less plasmid for each ligation, 0.1-0.5ul of plasmid instead of the 1ul reccomomended, extends the life of the kit however you just need to buy extra ligase which is next to nix compared to the cost of the kit.

good luck

Nick

Thanks Nick

I was told by the tech support at invitrogen that one reason why my TOPO TA cloning may have failed was because I had 2.5ul of product (230bp) which was way in access of the 3:1 molar ratio.
Since no where in the Manual do they mention using amounts equivalent to the 3:1 molar ratio, I was wondering how regualrly do folks make these calculations?