Blunt-end/cohesive ends cloning - (Dec/14/2007 )

I'm doing a sub/cloning experiment where I want to transfer the CDS of my interest protein from a puc plasmid to pegfp/c1. The problem is that I can only use HINDIII as enzime that originate a cohesive end, all the others from the MCS also cut inside the proteine sequence.
So i was trying to do it by amplifying the fragment by PCR, using A Forward primer with the hind III site, and R without any. I cut the plasmid with himdIII and SmaI (blunt end) and then Ligate.
Till now didn't work, but my fragment is 3kb, almost same size as the vector, and I'm using the Fermentas Rapid ligation kit. I tried already several different inbubation times, missing trying temperatures.

Can you give some help tips how to make it work>
Thanks

Are you amplifying your DNA with a normal Taq or a proof-reading? If it's a normal one then you'll have A-overhangs and the SmaI cut won't be of any help.

how many bp do you have skirting the HindIII sites on your PCR insert? Just checking to make sure things are okay

Are you sure you can't use any of the restriction sites in the MCS? What sites do you have in the MCS? And what sites do you have in the insert?

You could also compatible ends

BglII - BamHI - BclI all produces ends that are compatible with each other
SalI - XhoI both produce compatible ends
NheI - SpeI - XbaI all produce compatible ends

When partiall filling the ends

The BglII-BamHI-BclI group becomes compatible with the SalI-XhoI groups

Not pfil becomes compatible with XmaI pfil

There must be atleast one enzyme that doesn't cut in your gene. The best would be to amplify and have some site in the reverse primer. There are lots of good enzymes that might not be in the gene.

Age I, apaI , bsRG I, bstB I, cla I, Mfe I, Mlu I (can be tricky at times but works), Pspom I.

these are some enzymes (mostly not in MCS) which I have used with good results.

I have 4 bp, that I Think it's enough.
I checked for all the RE suggested by the manufacturer in the MCS and a few other one. all of the enzymes you told me cut also inside the insert
Thanks

okay.

Just to confirm matter, a proof reading polymerase is being used right?

Hmmm..... how bp saperates the SmaI and HindIII resistiction site on the vector? Is it sufficient for both enzymes to cut without problems? SmaI also cuts at 25 Celsius... lower then usual temperature.

Are the various fragments gel purified? Do be careful with the UV light, over exposure will cause cross linking reducing the transformation effiency of the DNA. Is dephosphorylation being conducted? It is not required here.

How is the ligase, have others in your lab experienced any problems with the ligases? T4 ligase does go off rather easily. T4 ligase buffer goes off too although less quickly.

How much DNA are you ligating? Do you have enough DNA to conduct a test.... ie after the ligation, and prior to transformation run some of the ligated DNA onto gel (use a narrow comb to help visualise what little DNA is there). With ligated DNA, you should see bands larger then your vector.

okay.

Just to confirm matter, a proof reading polymerase is being used right?

Yes

Hmmm..... how bp saperates the SmaI and HindIII resistiction site on the vector? Is it sufficient for both enzymes to cut without problems? SmaI also cuts at 25 Celsius... lower then usual temperature.
Because of this temperature problem I'm making a two steps digestion. Restricion sites are sperated by 30 bp
Are the various fragments gel purified? Do be careful with the UV light, over exposure will cause cross linking reducing the transformation effiency of the DNA. Is dephosphorylation being conducted? It is not required here.
negative for both answers. I'm cleaning it though between each digestion, but with a kit
How is the ligase, have others in your lab experienced any problems with the ligases? T4 ligase does go off rather easily. T4 ligase buffer goes off too although less quickly.
i'm the only one performing it right now

How much DNA are you ligating? Do you have enough DNA to conduct a test.... ie after the ligation, and prior to transformation run some of the ligated DNA onto gel (use a narrow comb to help visualise what little DNA is there). With ligated DNA, you should see bands larger then your vector.
I'm using the amount recomended between 50 and 100, i'll try to do it

Thanks a lot