I have a 1.4 kb insert flanked by Kpn-EcoR1 that I am attempting to ligate into a pCDNA backbone. Up to this point, I have gotten no positive clones. I have done successful transformations with uncut vectors. My ligation protocol has worked for previous cloning experiments. I have also sequenced my primers to make sure they are adding the appropriate restriction tails.
Prior to ligation, I ran about 2 uL of gel purified digested insert on a gel to determine roughly how much material I have. The band is pretty faint but I can still see it on gel which leads me to believe that this is enough for ligation (I typically use 8 uL of insert + 2 uL of vector).
I have also tried blue-white selection. But, my sequenced white colonies don't possess the insert.
Any thoughts? Should I try to re-amplify my PCR product to get more product? I am using Phusion so mutations are not as big of a concern.
Hi Sushanth,
If you see your band on the gel, I wouldn't worry too much as it is very likely you have enough to do your ligation. It would be more helpful to provide the ligation ratio, and amount of DNA you have used.
I normally tend to work out my DNA concentrations based on 1:3 ratio (vector:insert) and I use only 50 ng of the vector.
Also, you can dephosphorylate your vector to reduce your background colonies.
Good luck
Thanks Rajaei,
I can give a rough estimate on the amount of vector that I am using but am not sure about the insert. I digest about 1 ug of vector in a volume of 30 uL (33 ng/uL). I gel purify the digested vector (Qiagen) and then use 2 uL for ligation. I am assuming that the ligation mixture has about 50-60 ng of vector. For my insert, I take my initial PCR product, PCR purify it, then digest all of it. I run this out on a gel and then gel purify and use 8 uL for ligation.
For some reason, the Qiagen gel purified samples don't provide very clean samples on our nanodrop so I cannot give exact quantities. For reference, my insert is 1.4 kb and vector is 5.4 kb. I don't know how much playing with the ligation ratios will let me go from 0 colonies to >0 colonies. It seems like that is more of a question of efficiency not the reason I am getting nothing.
Also worth noting that I get 0 background colonies. I have repeated this several times as well. I have gotten background colonies before when I digest with different restriction enzymes but not with Kpn and EcoR1. I am not sure how to interpret this other than the fact that my vector is not religating.
I didn't mention this but I have also tried overnight ligations with T4 ligase and still no luck.
Thanks for the response Cornelius,
The spectrum just doesn't look clean and the A260/280 ranges anywhere from 2.1-3. The reported yields on the nanodrop are also very low for gel extracted fragments (even if I know that I have plenty of DNA).
I suppose I could compare to a marker to get a rough estimate.
Thanks for the tip on TA cloning, I will look into it.
may be your insert is toxic for the bacterium, try a low copy plasmid?
Hi Herve,
Interesting suggestion. The insert in question is part of a larger construct which transforms just fine. I am just trying to PCR out the insert and add restriction enzyme sites at the ends for some other subcloning. I find it unlikely that toxicity is the issue but it is worth considering.
Also, it might be a naive question but what benefit would TA cloning have over cutting pBluescript with EcoRV (blunt cutter) and directly ligating a Phusion generated PCR product (this is what I have also tried but with no success).
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