I am subcloning a gene present in a TA vector into an expression plasmid. This process involves the following;
- Digest of the TA vector containing the gene of interest with Xba1 and BamH1. Also, digest the expression plasmid with Xbal and BamH1
- Run the digestion reaction on 1% agarose gel and excise the precise bands (insert and digested vector)
- Extract the respective DNA (insert and vector) using a gel DNA extraction kit (Monarch's)
- Perform a Ligation reaction of the eluted insert according to NEB protocol with T4 Ligase.
- After ligation, I proceed to transform in XL10 competent cell but I do not see colonies afterwards
Recently did this, and found 2 colonies and when I analyzed the colonies, they did not seem to have my vector construct containing my gene of interest. As a matter of fact the plasmid I saw had a higher band size than expected.
At this point, I am not sure what/ where the problem is coming from. I would sincerely crave any suggestions or solutions to making this subclone successfull.
Thank you Dr. Shin, I used 20ng/ul vector (5.8kb) and 15.5ng/ul insert (1.5kb) that is in a 3:1 molar ratio of Insert to vector. I used the NEB ligation calculator to do this.
Hi,
did you do a control for the ligation and the transformation? I recommend for the ligation procedure to set up one additional reaction lacking the insert and another one lacking insert and ligase. For the transformation you may also use an undigested vector just to check if the transformation was successful and the section marker works as expected.
Good luck!
As Thomas Svoboda pointed out, you need to have the appropriate controls. The fact that you only got 2 colonies is problematic, but is it because your transformation did not work, or the ligation did not work, or that you didn't really have enough DNA. First I would be sure you have transformation controls, if you use uncut plasmid and transform, what do you get? The plate should have not hundreds, but rather hundreds of thousands of colonies. If not, the frequency is too low.
Secondly the ratio of vector to insert is fine, but your DNA concentrations are on the low side. When you state your vector is 20ng/ul is that the final concentration in the ligation mix, or the concentration of the vector that you then add 1ul into a 10 or 20ul ligation mix?
Lastly you can do some controls with the ligase and ligase buffer and be sure they are working well. For example you can use a single digested plasmid and show that relegated plasmid will give you LOTS more colonies after transformation than the non-ligated control.
Control experiments are your best and only friend when something doesn't work.
Thank you so much @ Michael J. Benedik.
My vector concentration after cut and gel DNA extraction was 20ng/ul (in a total volume of 10ul) and I used 1ul of it in the ligation. My insert was 8ng/ul (in 10ul total) after I cut out of the TA vector. I used 15ng of insert (2ul) in ligation reaction.
Thank you Thomas Svoboda I usually have an undigested plasmid control when I do transformation. I usually see a lot of colonies with that. Perhaps I will need to check my ligation system is working by using a religated vector transformation control.
Thank you Jong-hwan Shin. Would you recommend using a PCR machine or an incubator to set up the ligation reaction at 16C?
Another thing that comes to my mind is that probably one of your enzymes do not work. You could try single digest of a plasmid with one enzyme respectively using the buffer you take for your other digest. On the gel you should see clearly if the plasmid was linearized or not. Load digested/non digested plasmid side by side.
Thank you Thomas Svoboda I have done a test on my digestion enzymes on the vector that contains my insert and on the expression plasmid and both cut well
I'd suggest running the undigested TA vector and expression plasmid as controls on your gel (to check for digestion of both the gene of interest and expression plasmid), doing a range of insert:vector ratios for ligation (3:1 is standard, but I've also done 1:10 and 10:1 in the past and it helped a lot) and transforming with undigested expression plasmid to check your cell transformation efficiency. If you haven't already, I'd also check the exact transformation conditions of XL10; I've run afoul of transforming cells because I assumed all protocols were the same!
Good luck!
To put it simply, I think the reason, why you aren't getting the desired clone despite of getting through the check points you mentioned, is somewhat lies in your transformation into competent cells. Here again two factors, either your comptent cells are low in efficiency or you did something wrong on transformation front.
Solution to your problem is that just redo your transformation step with a positive control plate.
@Hugh White, i have checked for digestion and transformation efficacy. I usually run undigested TA vector on gel alongside the digeated one. You can see a clear cut of the digested gene of interest. Suggesting the enzymes are working. Also i checked for transformation efficacy of XL10 using undigested plasmid. I saw lots of colonies which tells me that the transformation with the competent cell is not the issue. I haven’t titrated the ligation conditions using different ratios, i use the 3:1 of insert to vector. Do you think usung diffferent ratios will help?
@akraam ali, i did control of my transformation conditions with only the expression plasmids and saw lots of colonies.
@Somtochukwu Stella Onwah, I can't say for sure, but it helped when I did a ligation.
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