9 replies to this topic

[nIUro]

I'm having some troubles cloning into a plasmid that I've doubly digested with BamHI and NotI. The distance between these 2 restriction sites is 19 bp. I'm trying to ligate in a test fragment made of 2 x 30bp hybridized synthetic oligos, but I'm not getting any colonies when I transform the ligation into competent cells. I'm also not getting any colonies when I transform the cut plasmid only.

The digest was done with 5ug of plasmid, 3ul BamHI, 6ul NotI, 20ul NEB #3. Digested at 37 overnight, gel extracted. Single digests using each enzyme individually show that it does cut the vector, and only once.

The oligos were prepared by mixing 0.5 uM of the 2 oligos, 180 mM NaCl, 50mM Tris. Incubated at 60C for 5 min, 50C for 10 min.

The ligations were done using 40ng of doubly cut vector, 1ul T4 Ligase, 1ul 10X Buffer, 1ul oligo. Incubated at 14C for 3 hours, then 4C overnight.

Transformed into NEB Turbo cells. No colonies.

So any suggestions? This cloning has been pain!

[phage434]

Are the oligos phophorylated?  They need to be to allow ligation.

[perneseblue]

nIUro, on Feb 17 2009, 08:17 AM, said:

The digest was done with 5ug of plasmid, 3ul BamHI, 6ul NotI, 20ul NEB #3. Digested at 37

Just checking. Does this mean the total volume of the digest was 200ul? Given that this is an overnight digest, you don't need to use so much enzyme. (unless the volume of enzyme quoted has been diluted). 1.5ul BamHI and 1.5ul NotI should be enough.

Just checking. When the oligoes anneal to each other they do produce a 5' overhang? Was it treated with polynucleotide kinase PNK? As mentioned by phage434 the oligos should be 5'phosphorylated

[nIUro]

phage434, on Feb 17 2009, 11:51 AM, said:

Are the oligos phophorylated?  They need to be to allow ligation.

Maybe I'm blanking on my molecular biology, but I thought restriction enzymes leave the 5' end of the plasmid phosphorylated, which is why I didn't bother to phosphorylate the oligo insert.

[nIUro]

perneseblue, on Feb 17 2009, 12:07 PM, said:

nIUro, on Feb 17 2009, 08:17 AM, said:

The digest was done with 5ug of plasmid, 3ul BamHI, 6ul NotI, 20ul NEB #3. Digested at 37

Just checking. Does this mean the total volume of the digest was 200ul? Given that this is an overnight digest, you don't need to use so much enzyme. (unless the volume of enzyme quoted has been diluted). 1.5ul BamHI and 1.5ul NotI should be enough.

Just checking. When the oligoes anneal to each other they do produce a 5' overhang? Was it treated with polynucleotide kinase PNK? As mentioned by phage434 the oligos should be 5'phosphorylated

Yeah, the total volume was 200ul. Would the amount of enzyme that I was using result in star activity?

[perneseblue]

nIUro, on Feb 17 2009, 09:15 AM, said:

Yeah, the total volume was 200ul. Would the amount of enzyme that I was using result in star activity?

Given the volume used, star activity would not be expected.

PS. ah , my mistake. The vector was not dephosphorylated. So it would be fine.

[T C]

Hello

Point 4 is the most relevent.

1.  Bam HI is tricky and doesn't work that well with double digestions (atleast in my hands).  With BamHI, I always go for sequential digestions if its a preparative digest.

2. Since the sites are close, CIP it to prevent religation of singly cut vector (though u don't see any colony in yr case).  Alternatively, use a clone with these sites so that you can easily distinguish between single cuts and doubly cut vector.

3. You need to phosphorylate yr oligos or the PCR product if you go in for blunt end ligation but not if you are digesting teh PCR product b4 ligation.

4. I think you are not seeing colonies as yr vector size is huge,  try precipitating the vector after digestion, it has always helped me when i clone large fragments.  Running on agarose somehow inhibits ligation (personal observation).

Best

TC

[stardust]

Hi there,

I just wanted to add that I have a different protocol for oligo annealing:

1 µg per oligo, same buffer as you in 20 µl total

Incubate at 95°C for 5 min in a pcr cycler, let cool to room temperature in the PCR cycler for about 2 - 3 h, check annealing on 3 % agarose gel

then use 100 ng vector and 2 µl of the annealed oligos for ligation

Maybe that helps?

Stardust

[Nrelo]

T C, on Feb 17 2009, 10:40 PM, said:

Hello

Point 4 is the most relevent.

1.  Bam HI is tricky and doesn't work that well with double digestions (atleast in my hands).  With BamHI, I always go for sequential digestions if its a preparative digest.

2. Since the sites are close, CIP it to prevent religation of singly cut vector (though u don't see any colony in yr case).  Alternatively, use a clone with these sites so that you can easily distinguish between single cuts and doubly cut vector.

3. You need to phosphorylate yr oligos or the PCR product if you go in for blunt end ligation but not if you are digesting teh PCR product b4 ligation.

4. I think you are not seeing colonies as yr vector size is huge,  try precipitating the vector after digestion, it has always helped me when i clone large fragments.  Running on agarose somehow inhibits ligation (personal observation).

Best

TC

Same experience as point 1. BamHI does not work as good as other RE (EcoRI, HindIII) in double digestion. I usually double digest the vector/insert in a sequential manner when BamHI is used.

[phage434]

You might want to try the new BamHI-HF high fidelity version of BamHI from NEB, with reduced star activity and more compatible buffer conditions.  There is also an EcoRI-HF enzyme which we have been very pleased with.