8 replies to this topic

[philman]

I am attempting a transformation at the moment of a 1.8kb insert into a 9kb lentivector

I have cut with my relevent enzymes (BclI on the insert and BamHI on the vector, it is the same site either side of the insert so I will hve to check later for transformants the correct way round, and BclI and BamHI have the same overhang so should ligate) for 4 hours each, I have added TSAP Alkaline Phosphatase to the vector for the last 5 mins of the digestion, I gel purified the DNA, I did a ligation at 4 degrees overnight then plated onto Amp plates.

I am still getting loads of colonies on my control plates, both vector alone plus ligase, and cut vector alone without any ligase!

I thought it might be due to uncut vector contaminating samples but I digested for a long amount of time, although when I ran the cut vector on the gel I did notice it was a funny colour and I think some of the water bath water may have leaked into the tube while the DNA was being digested, but the band on the gel seemed very distinct so I wasn't that worried about it inhibiting the digestion, mabe i hould have been though.

[ElHo]

Maybe you have a contamination problem. Did you also perform a negative control, i. e. transform only water?

[philman]

no I didn't this time, but I did when I used the same batch of competent cells last week and got zero colonies the day after (although I did leave the plate in the incubator over the weekend out of curiosity and came in to find colonies on Monday, 4-5 days later, which weren't there the day after transfoamtion)

I also did a count on the plates, with about 75 colonies on the cut vector without ligase plate, and over 300 or so on the other plates (vector+insert+ligase and Vector+ligase alone) Which does imply it is a ligation problem as well.

[perneseblue]

Right....

It is good that you have checked  the competent cells for contamination. Since you see no colonies after 16hr of incubation, I would say the competent cells are clean. Antibiotics are heat labile, so incubating the plate for 4-5 days would destroy the antibiotics allowing sensitive cells to resume growth and thus produce the colonies.

Given the assumption that the competent cells are clean.
I believe there are a few problem with here.

A major problem appear to be uncut vector contamination. This is represented by the 75 colonies that you see with the vector + no ligase plate. (1/4 of all colonies recovered) The way to remove this problem is to gel purify the vector DNA. You should also consider a longer digest next time.

The next problem appear to be incomplete vector dephosphorylation. Thus the ~300 colonies detected on the vector+ ligase plate. There two ways to fix this problem.

1- dephosphorylate longer. I use antarctic phosphotase. It is more forgiving and allows heat inactivation. If you use CIP (which you aren't) be very careful as over dephosphorylation will render your vector unligatable.

2- Try using a different ligation strategy. At present, the vector can re-circluarise as both ends of the vector are compatible with each other (both BamHI overhangs). If you cut the vector with two RE, which incompatible overhangs, the probability of vector religation is reduced immensely. Some people don't dephos their vector has been cut by two RE.

[philman]

yes I am doing it again now with a much longer digest time, and I was gel purifying to begin with so I guess the contamination was in the band I cut out for some reason. I realised that when I saw all those colonies, but at least I know that my ligase is working since I got so much more on my +ligase plates. And I know I need to use much lower volume for each plate!

I am using TSAP from Promega at the moment which is also heat inactivable but this doesn't seem to matter for my protocal as I just stick the digestion plus TSAP straight into the gel after the incubation is up! I stick 0.5ul of TSAP into my digest and stick it back into the 37 degree water bath for 15 minutes, then remove it 15 mins later, take it over to the gel, add the loading dye and run it. Should I add the TSAP earlier?

I know the cut ends with BamHI can religate, and the TSAP was supposed to get around that! if this still fails this time then I will order new primers, the vector also has a XhoI site I could use

[GNANA]

i guess the prob might be the phosphatase, if poss try to get CIP jus for a reaction (1 micro litre) anywhere and try once before you go for a primer change,
i have done lot of clonings using CIP, i usually do max 1 hr incubation at 37...i always got nil colonies in vector alone plate...

Another problem might be in Restriction digestion of ur vector, after digestion run bit longer time as u have to completely seperate the digested vector from uncut if any, then u proceed for gel extraction.

good luck,

gnana...

[perneseblue]

philman, on 17 September 2010 - 01:17 AM, said:

TSAP straight into the gel after the incubation is up! I stick 0.5ul of TSAP into my digest and stick it back into the 37 degree water bath for 15 minutes, then remove it 15 mins later, take it over to the gel, add the loading dye and run it. Should I add the TSAP earlier?

Sounds okay. Looks like you have things under control. Best of luck

[wincel]

If your original plasmid has inside of the removed sequence a restriction site that is not present in the ligated plasmid, re-digest the plasmid with that restriction enzyme for 30 minutes after the ligation.
But make sure you heat inactivate this or it will decrease the transformation efficiency.
You can just add the enzyme into the T4 DNA ligase buffer, pretty much any more frequent used restriction enzymes digests in this buffer.

That way your re-ligated or uncut plasmids are linearized and will not yield useful colonies decreasing the background colony number tremendously.
This works much better in my hands than dephosphorylation of the vector backbone or gel purification. I skip both of the later ones in case there is a useful restriction enzyme for re-digest after ligation.

Edited by wincel, 24 September 2010 - 02:19 PM.

[philman]

wincel, on 24 September 2010 - 02:18 PM, said:

If your original plasmid has inside of the removed sequence a restriction site that is not present in the ligated plasmid, re-digest the plasmid with that restriction enzyme for 30 minutes after the ligation.
But make sure you heat inactivate this or it will decrease the transformation efficiency.
You can just add the enzyme into the T4 DNA ligase buffer, pretty much any more frequent used restriction enzymes digests in this buffer.

That way your re-ligated or uncut plasmids are linearized and will not yield useful colonies decreasing the background colony number tremendously.
This works much better in my hands than dephosphorylation of the vector backbone or gel purification. I skip both of the later ones in case there is a useful restriction enzyme for re-digest after ligation.

That would have been a brilliant way to do things, but the restriction site I could have used (BamHI) by pure coincidence happens to be present right in the centre of the sequence of the gene I am trying to clone into the plasmid!

Still I have got them working now I think Got colonies and positive results on the colony-PCR, as well as on the digests, last week. Just going to sequence them this week to make sure

Thanks for all your help!

Edited by philman, 27 September 2010 - 06:14 AM.