Guys I need help please - (Jan/27/2006 )

hey guys,

I'm doing 3 fragments ligation. I was able to digest the vector with the required restriction sites AfeI and XbaI, but I lost one bp form my open reading frame at the afe site.
also, I was able to digst one of the fragments with xmaI and xbaI perfectly. so, the remaining fragment is the one with afeI and xmaI. in fact, I don't have these restriction sites in my fregment. therefore, I used PCR to introduce these sites as well as the lost bp and here is my problem.
after PCR amplification, I got the perfect band size. but since it's 3 fragment ligation and there are too many variables, I tried to subclone this PCR product into TA vector. Everything went perfect I got perfect colonies. PCR screening with primers on the vector gave me bands with the required size, but I was not able to digest them at all. I sequenced them and I got strange sequences at the both terminals. I don't know from where.

it has been 3 months now and nothing happened. so, could you please give me any hint or alternative way to do my cloning please.

regrads,

Hi, I like your plan, would have suggested subcloning into TA.... When you say bad seq at both termini, are you sequencing through?? What I mean is it is difficult to get really good sequence from the ends of the insert with your typical T7 and SP6 b/c takes a certain number of bases to get the sequence reaction going well so you get bad sequence close to the sequencing primers...

other than that will you post your primers, and give some info like how many bases long, how close to the end are your engineered RE sites etc...

are you subcloning into T-easy?? maybe an alternative is to not add the RE site with your primers and use the T-easy MCS?? (have to check the map to see if your sites are available...)

HTH

thanks alot,

regarding your questions, yea I'm sequencing all the fragment. Actually, I'm subcloning my fragment into PCR2.1 vector (invitrogen). I'm using M13 forward and reverse primers which are on the vector as well as different primers on the fragment.
these are the primers I'm using to introduce the restriction sites:
Forward 5'-AGC TTC AGC GCT TAT GGC GTC TCA AGG CAC C-3' (31 bases)
Reverse 5'-TGA CGT CCC GGG GCC ACC ATT GTC ATA TTC CTC TGC-3' (36 bases)

as you see, the blue bases are on the fragment while the red ones are the restriction sites. The black bases in between are needed to correct the open reading frame (T in the forward primer is the lost base I told you about) and I need those two a.a on the reverse primer between my fragment and the other one I'm gonna ligate to.


back to sequencing, I'm getting perfect sequence aligned with 100% homology with my fragment, but after that I mean at both ends I got very strange sequences (varies from 10 to 50 bases) I don't know from where. after these strange sequences I'm getting perfect alignment with the TA vector (PCR 2.1) although they are not from the beginning expected on the vector. that's why I'm think of that the problem maybe in the PCR itself. the added sequences are not being introduced, instead these strange sequences are introduced.

now. I'm gonna sequence my PCR products before transforming my comptent cells.

man, any idea

I would guess that the primer was made incorrectly. This happens sometimes. Reorder the primer and go again.

You might want to add the optimal TA cloning overhang 5' of your restriction site,
GTTTCT, which will assist you in Topo-TA cloning:

M. J. Brownstein, J. D. Carpten, and J. R. Smith. Modulation of non-templated nucleotide addition by taq dna polymerase: primer modifications that facilitate genotyping. Biotechniques, 20(6):1004–6, 1008–10, 1996. PMID 8780871.

thanks phage,

I thought about that and I ordred them with OPC purification (95% purity as the company said)
and I got no digestion again...

actuallt, I'm getting frustrated

do you mean what I added is not gonna work??

PCR2.1 is a TOPO TA vector?? I would agree with phage, but it would be unusual for both of your primers to be made incorrectly, I never had one primer made wrong (ordered from sigma and Gibco) although they could have had a really bad day...

I don't think that even with TOPO system having the optimal sequence is a requirement, it seems like your cloning is working ok so while it may improve efficiency or something I wouldn't worry about it unless you are using a TOPO system and you do order new primers, then I would follow phage434 advice and change the 5' end bases to the most optimal ones for TOPO cloning...

I never used a TOPO vector, always did TA into pGEM T-easy.... Maybe something about this enzyme???