cloning problem ....... - (Jul/25/2007 )

Hello everyone...
I am doing cloning 2 kb pcr produt....
while transformation i am getting white colonies but no positive clones found after many transformations....

I want to tell u about dna concentration...
after pcr amplification of 2 kb dna is very faint is this the reason for failure...?

i am trying this from 1 month...plz help me...


Thanks

Hi Santosh,

When you have a faint band any PCR clean up protocol will result in further loss of DNA. Is there any way you can scale up the reaction or do a second round of PCR to increase the amount of starting material?

Cheers,

Scott

i don't very agree to scott proposal as i think that second round of amplification raise unspecific events.
1st : do you gel purify ? try to avoid this step.
second, is it a TA cloning kit or similar ? in that case do you add extra Atail with Taq ?

could you not try to improve the PCR reaction yields? What polymerase are you using? Where does you template DNA come from? Is it genomic or plasmid/BAC/PAC? Have tried optimising the annealing time? Or MgCl2 or KCl concentration? Tried betaine, or 10%glycerol or BSA?

Thanks to all
i tried every ways u have suggested now...
and many of my friends suggested same way like scott.......
so i got today only bright band after 2nd pcr...
so lets see this time ligation will work or not........

thanks to all again.....
all d best
cheers.......