Poor ligation efficiency using Invitrogen TOP10 and XLI-blue cells - (May/26/2008 )
Hi guys, I recently tried to do a control ligation by chemically transforming some Invitrogen TOP10 one shot and lab-made XLI-blue cells with a plasmid on a pUC 19 background and no insert. This plasmid was first digested with Mlu I, then ligated together for 2 hours at room temp. The concentration of plasmid I used was about 100ng/uL, using 2.5 uL of plasmid or 0.25 uL of plasmid with 0.5 uL T4 ligase and 0.5 uL 10X ligase buffer, topped up to 5 uL total volume with deionized water.
My PI tells me that I should be getting massive amounts of colonies, since I didn't alkaline phosphatase the plasmids before ligation, but I'm only getting ~150 colonies with the 2.5 uL plasmid on the XLI-blue cells (no colonies with 0.25 uL plasmid), and 2 and 3 colonies with 2.5 uL and 0.25 uL plasmid respectively using TOP10 cells. I have no idea why this is the case. We just bought new ligase, so this shouldn't be the problem.
Prevously, I had trouble getting any colonies at all when I did the same ligation and transformation, except with phosphatased plasmids.
Does anyone know what the deal is with the low efficiency??
Alex
My PI tells me that I should be getting massive amounts of colonies, since I didn't alkaline phosphatase the plasmids before ligation, but I'm only getting ~150 colonies with the 2.5 uL plasmid on the XLI-blue cells (no colonies with 0.25 uL plasmid), and 2 and 3 colonies with 2.5 uL and 0.25 uL plasmid respectively using TOP10 cells. I have no idea why this is the case. We just bought new ligase, so this shouldn't be the problem.
Prevously, I had trouble getting any colonies at all when I did the same ligation and transformation, except with phosphatased plasmids.
Does anyone know what the deal is with the low efficiency??
Alex
It depends on a lot of things why you are having low transformation efficiencies:
1) Too much DNA:
If your pUC DNA is relatively high, you could dilute it in TE buffer, and use less.
2) Try electroporation. I've used pUC18 (same as pUC19 but the MCS is reversed) and XL-1 blue as a control. I used 1uL of 10 picograms/uL pUC18 DNA, and I got somewhere around something x10^9
The first thing you should do is to test the transformation efficiency of your cells. Until you are getting repeatable results from your transformation, I would recommend always doing a control transformation with 1 ul of pUC19 plasmid (uncut) at 10 pg/ul. You should get hundreds of colonies (1e9 colonies/ug * 1e-5 ug/10 pg * 10 pg = 1e4 colonies). Debug this problem first. Possible problems include too much volume, too short/long/wrong temperature heat shock, wrong medium for recovery, mistreatment of cells prior to transformation. The Top10 cells come with a vial of 10 pg/ul pUC19 DNA for this test.
Hi phage434, thanks for your reply. In fact, I did the test you suggested at the same time I did the transformation with my plasmid. I got an efficiency of about 5x10^8 colonies/ug DNA, so I don't think it's a problem with the efficiency.
As for the concentration of DNA, I thought that the more DNA you put in, the more cells you were supposed to get but I got a pretty low number even though my DNA concentration was really high. Unfortunately, I don't have an electroporation machine nor do I have electroporation competent cells. I'm going to try the ligation with my insert tomorrow, hopefully I get some colonies with my insert in it.
Alex
OK, I agree with your PI. Please give more detail on your digestion and ligation reactions (components and volumes, amounts of DNA, heating, purification). Are you heat killing the RE prior to ligation?
What is heat killing?
Just kidding
Yes, I did heat kill the RE, plus I did a gel extraction using the Invitrogen gel extraction kit to cut out my band before I did the ligation. The band looked like it was completely digested (linearized) when compared to the undigested plasmid I ran at the same time, and it corresponded to the correct band size, so it's likely not a problem with the restriction digest.I talked to him again this morning, and we're thinking it's something to do with the ligation for sure. Here's what I used:
3.75 uL MilliQ H2O
0.5 uL 10x ligation buffer
0.25 uL plasmid DNA
0.5 uL T4 ligase (2,000,000 units/mL)
and
1.5 uL MilliQ H2O
0.5 uL 10x ligation buffer
2.5 uL plasmid DNA
0.5 uL T4 ligase (2,000,000 units/mL)
For the XLI-blue cells, they came in 200 uL aliquots from the -70 C. I unfroze them on ice, and added my ligation reaction and incubated on ice for 30 minutes. Then I took them and heatshocked them for 60 seconds at 42 C, iced them for 10 minutes, and added 400 uL LB broth. This concoction was centrifuged, and I removed 500 uL of supernatant then resuspended in the remaining 100 uL. After that, I plated the entire 100 uL on LB-amp plates (100 ug/mL).
For the TOP10 cells, I basically followed the manufacturer's protocol which called for almost the same procedure as above, except they were heatshocked for 30 seconds, and I incubated the TOP10 cells in SOC medium for 60 minutes at 37 C before plating 100 uL (total volume of 300 uL).
Hopefully something works today (fingers crossed).
Alex
