cloning blues! - (Nov/27/2011 )

i have beeen tring to clone a 4.2 kb product in pGEM T easy vector for past 6 months all in vein ..

i ll brief in short all that i have tried

i standardised the pcr product . and purified it . then quantified and set up the ligation reaction in 1:1, 1:2, 1: 3 vector insert ratio and set it.
then transformed it in competant cells DH5alpha nd plated on lb + amp and incubated for overnight .

pin point colonies appear but they are invariably empty vectors and wen PCR is done unspecific band is seen.

i thought may be the vector has lost terminal t therefore made a control reaction with vector only but no colonies appeared.

i am not assuming that the cells are not working cause other clones came out well.

i also tried the cloning with TOPO XL kit .. the prob arrises there too.
Enpty vector having unspecific insert.

I thought may be there is some contamination therefore i ran purified product on gel and gel purified it again. but still the problem persists.

my guide asked me to use new competant cells BL21 and also to try blunt end cloning ..
but can anyone have any ideas wats going wrong ?

i m clueless right now. thanks for your suggestions.

oh i also did ligation at 4 and 16 degree celsius... anything that u guys can think of please let me know . i m really stuck here

You got to concentrate on the following,

Try to ligate the pcr product without purification - sometimes on purification the terminal A would be lost, in this case you got to optimise the PCR for the specific product.
check the molar ratio of pcr and vector on gel. Increase PCR input further more.
check whether your polymerase has the terminal transferase activity.
Above all your competent cells should be enough efficient to take up and process the Vector with insert. so check the competency of the cells you are using.

goodluck...

TA cloning is not very selective about what it clones. I would recommend switching to conventional cloning with 5' overhang restriction sites (different on each end) and vector cut with the same two enzymes. Don't forget the 5' extra bases (six for safety) beyond your restriction sites.

I'm suspicious about your sequencing results. Perhaps you really have your clone, and simply don't know it. How are you preparing DNA and then sequencing? What primers are you using? Can you verify the plasmid in these colonies with restriction cutting? Or with PCR (one primer on the insert, another on the vector)? What size is your transformed vector?

I've also tried a 1:10 ratio, which helped me push through some difficult cloning at times. Crude, but it might help you

GNANA : I tried to standardise the PCR a lot but the primer is giving a very bright band at 1500 band and no matter how much i changing mg and temp that jus not goes.
i am using advantage taq from clonetech that has terminal transferase activity so that should be a prob.

phage 434: i have restriction enzyme sites in both forward and reverse primer they are sac 2 and kpn 1 . but i don have both these sites in pSK+ or pGEM t Easy vector. nonoe of the lones have showed no difference in migration pattern thats y i didnt give it for cloning yet. they have similar band pattern to empty vector . i have tried checking with restriction digestion and pcr also . but no satisfactory results come.

i :10 insert vectr ratio?
or vice versa?

thanks guys for your valuable suggestion if u can think of anything else please do let me know.

i also thought may be exposing to uv during purification is causing prob thats y did purification with SYBR green in blue light .
also , going to do blunt end ligation .. for that i m using Pfu polymerase.
gonna cut pSK+ with eco R5 and try to do that .. i m sure its gonna be even more tough but i m left with no choice.

if you couldnt avert non specific amplification, do a second round of PCR on gel purified product and see whether you could attain the specific product, if you get use this for downstream cloning.

You could amplify the vector with sacII and KpnI restriction sites in the 5' end of the primers. Then, in the PCR buffer, cut with DpnI to eliminate template circular DNA, purify, and mix with your purified PCR product (uncut) in appropriate ratios. Cut with sacII and KpnI, purify (you can't heat kill KpnI) and ligate. This sounds like a long path, but is actually quite easy, and provides a way of cloning any restriction enzyme site into any vector (as long as the vector is not cut by the enzyme you are using).