ligation problem - (Mar/23/2009 )

hi..
Can anyone help me...

Im trying to make a probe for Citrus viroid(CVd) detection.

To achieve this, I designed primers with restriction sites added onto them, EcoRI (forward primer) and HindIII (reverse primer). Since CVd's presents themselves as RNA in the infected plants, I synthesized cDNA by reverse transcription polymerase chain reaction (RT-PCR), then RE digest the RT-PCR product with EcoRI and HindIII using Fermentas Fast Digest enzymes. Gel purify them using ZYmoGel Clean kit. and then quantify them using spectrophotometry, to get the concentration of the digested fragments. used the pGEM-3Z vector(~2743bp) (also EcoRI, HindIII digested, purified and quantified).

In ligation, used the Fermentas Rapid Ligation kit, as per manufactures recommendations . used 1:3 (vector: insert) concentration ratio (ug/ul) and molar ratio (pmole ends) @ 22°C for 1hour. heat inactivated T4-ligase @ 70°C for 5minutes. But when I run the ligation product on the gel (1% agarose gel), i don see any bands... Because I read somewhere that, sometimes you can not see the bands on the gel, I continued with the transformation, i used JM107 CaCl2 treated competent cells(Maniatis recipe) and fermentas Transform Aid bacterial transformation system.

Transformation
1) Fermentas transformAid competent cells i used 5ul ligation mix ( previously incubated on ice for 2minutes) and 50ul competent cells. incubated this mix on ice for 5 minutes. then platen on pre warmed 2xTY/ Amp(100ug/ml)/ IPTG/X-gal plates @ 37°C overnite.

2) for CaCl2 treated competent cells
5ul ligation mix (incubated on ice for 2 minutes)+ 50 ul competent cell. incubated on ice for 30 minutes. heat shocked @42°C for 90seconds (water bath) then put back on ice added glucose supplemented (100mg/ml) 2xTY broth (200ul) then shaken @ 37°C water bath for 1 hour and plated on 2xTY/Amp/X-gal/IPTG plates and incubated @37°C overnite.

my problem is on the control plates (pGEM-3Z uncut where i supposed to get a lot of blue colonies, i get a few blue and lots white colonies) and on other plates on good days i get 2-5 (very few) white colonies. which dont grow when re-inoculated on Amp/2xTY broth.

Does anyone know where can i make changes? or what is it that im not doing right? i've been struggling with this for 2years now... please help me.

Are you sure about the heat-shock step? 90 seconds sounds like a long time: I usually do 30 or 45 seconds, depending on the plastic.

mitogirl on Mar 23 2009, 01:53 PM said:

thanks, mitogirl will try reducing time. But the other other system (fermentas kit) does not need the heat shock step and that too does not work.

Try increasing the RE digestion and ligation time, possibly overnight. Also include a phosphatase step for the vector after RE, then heat inactivate enzyme, and ligate insert to vector. For competent cells, I usually only heat shock for 30 sec at 42C followed by 5min ice and 1hr at 37C with media prior to plating.
One possible check to do: After the RT-PCR, take ~2ul of the amplified PCR product and repeat the PCR procedure using your designed primers. This may sound silly, but it will be a good check to establish you have the correct PCR product. Plus, you can always use the excess PCR DNA for RE reactions.