Help needed with subcloning problem - (May/12/2011 )

Hi everyone,

I am having a crazy problem with a ligation that truly resist all my attempts so far. I do the whole thing and get no colony whatever i try.
I want to subclone a 2.7kb gene into a pCMV-Sport6 plasmid (around 4kb) into a pcDNA3.1 (around 5kb). I use KpnI (promega) and XbaI (fast digest from fermentas). I use the pcDNA3.1-CAT vector so CAT is removed from pcDNA3.1 after digestion by the two enzyme in order to check that both digestion worked. So here is what i do:
- digestion with KpnI 1h, 37°C
-DNA clean up using PCR clean up kit from invitrogen
- digestion with XbaI fast digest 20min, 37°C
-gel purification using Peqlab gel extraction kit
Bands were really clean and at the expected size for my gene to subclone, the digested pCMV-Sport6, CAT and digested pcDNA3.1 (amp resistance) . So i doubt that the problem comes from digestion.
During the cutting the UV exposition is about 20-30 second and unfortunately i do not know whether my transilluminator is low or high wavelength... I would rather say low but not sure.
Then after gel purification i ligated my gene to subclone of 2.7kb into digested pcDNA3.1 as follow :
-first i run a gel to assess the amount of DNA for the two digested fragment (my pcDNA3.1 digested vector was around 3 times more concentrated than insert)
-then i start ligation using insert/vector ratio of 1:1 and 3:1
- vector DNA: 2ul (10-20ng)
- T4 DNA ligase 5u (fermentas): 1ul (5u weiss)(maybe it is too much?)
- ligase buffer (aliquoted to avoid freeze-thaw cycle): 2ul
- insert: 6uL (1:1 ratio) or 12ul (3:1 ratio)

Than out of this i use 5ul of the ligation mix into 50ul of DH5a competent cells that i prepared myself recently. after heat shock of bacteria i add 200ul of LB and then incubate 1 hour than spread on ampicilline plate. I always use a transformation positive control usually pcDNA3.1 empty vector not digested. The positive control always give me loads of bacteria. My negative control is the selfligated pcDNA3.1 digested vector which gives no colony.
Despite the many tries that i gave so far i never managed to get even a single colony after transformation. I have read several similar topics and some people suggest the following ideas that i have not tried yet:
-avoiding UV exposition (which is my next thing to try)
-heating at 65°C the insert and vector DNA for 5-10min prior to ligation in order to avoid vector/vector and insert/insert ligation.
-adding 5% PEG to the ligation mix
-heat inactivation of ligase at 65°C after ligation prior transformation
-dephosphorylation of DNA

Does anyone think these suggestions could work? could it be a problem of size of insert compared to vector 2.7kb vs 5kb ?
I would really appreciate any suggestions because i am running out of solutions
In advance thanks a lot

Rémi

I know you get many transformants in your positive control, but you need to calculate a number for the CFU/ug of plasmid DNA. It is easy to make bad competent cells, ones that transform just fine with prepared plasmid DNA in large amounts. It is difficult to make highly competent cells. You need to dilute your plasmid positive control to the 10-100 pg/ul level using serial dilution, and then transform with 1 ul into your competent cells. Then count colonies and express the efficiency as CFU/microgram of DNA. Good efficiency is about 10^8 - 10^9 CFU/ug. I'm guessing you'll have more like 10^6, which means you won't see many transformants. 5 ul of ligation into 50 ul cells is too much -- use 1-2 ul.

phage434 on Thu May 12 17:42:58 2011 said:

As you guessed, the pUC19 plasmid gave me 10⁶CFU/ug with my competent cells. I have read that electro-competent cells were usually more effient than chemically competent cells, unfortunately, I do not have the material to make electro-competent cells. Would you have a good protocol to make more effient chemically competent DH5a cells?
Thanks a lot
Rémi

Hi there

XbaI is sensitive to methylation. It won't cut if the recognition sequence has a GA before it or a TC after it. ie: GATCTAGATC HAve you checked your sequence?

Clare

PS: Ooops ignore me - I didn't read your post thoroughly enough, obviously your digest is fine

Clare on Tue May 17 09:24:43 2011 said:

Hi there

XbaI is sensitive to methylation. It won't cut if the recognition sequence has a GA before it or a TC after it. ie: GATCTAGATC HAve you checked your sequence?

Clare

PS: Ooops ignore me - I didn't read your post thoroughly enough, obviously your digest is fine

Hi again,

just to finish up this post,
I got colonies !!!
I modified some steps especially at the ligation level and it went great:
- i did not expose the DNA to UV, i exposed a small amount of my digested DNA in a lane that i did not use for ligation
- i heated my DNA mix (with just the insert and vector but no ligase buffer/ligase) at 65°C for 5min as i read it was said to prevent DNA self ligation vector/vector and insert/insert.
-ligation was performed at ratio insert/vector 1:1 ; 2:1 and 3:1 but all of them worked at 16°C for an hour and then put at 4°C overnight.
-ligase was heat inactivated at 65°C for 10min.

My negative control (vector without insert) shows no colonies and ligation with insert shows between 10 and 20 colonies.

Thanks you all for support.

Rémi