Gateway cloning not working. - (Jan/13/2015 )

I am using BP clonase II and LR clonase II. entry vector is pENTR221. and Destination vector is pET-DEST55. I have 4 genes to clone and none of them are working. I have sequenced entry clones of all 4 genes in pENTR vector.

After performing LR cloning, I get colonies (TOP10 cells), I grow them in 2ml LB with carbenacillin, extract the plasmids and transform them into BL21 cells, I dont get any colonies there. BL21 cells are correct and competent enough, i have checked. plates are the same and working properly. other all things are proper i suppose. but still, somehow i am getting false positives after LR cloning step. I dont get any plasmid after growing cell in TOP10.

What could be the problem ? Help me oh Master!!!!

So, do you check that there is a plasmid in your transformed Top10 cells? That is step 1. You could restreak your colonies on a carb plate to double check that there is carb resistance. You should do a negative control of growing untransformed Top10 cells on the same media. Perhaps your Top10 cell stock is already resistant?

My TOP10 cells are ok and do not have resistance to carbenacillin. The peculiar thing is I get 2 type of colonies sometimes, large and small, according to Gateway troubleshooting guide, this is due to losing of plasmid due to large size or toxicity. My gene is neither large (1000bp) or toxic to cell (its beta lactamase).

when I extract the plasmid from TOP10 cells, the spectrometer shows values around 150ng/ul and purity of 1.6-1.8. but when I run the gel, the bands are very faint and does not correspond to conc obtained by spectro.

Also when I send the plasmid for sequencing, it shows empty insert. ie start codon - strep tagII - stop codon. 

it should be start codon - streptag II - My gene - stop codon.

which means, the chloramphenicol and ccdb gene in destination vector are recombined and gene is not inserted. thus the final product is empty.

How long do you grow your cells? The small cells sounds like satellite colonies rather than true transformants. Is your bla gene identical to the bla gene already present on the pET Dest55 vector? I could imagine some recombination issues if they are the same.

Hello,

the genes are different, but i tried a transformation yesterday with pDEST55 vector and my TOP10 cells, there were colonies present on the plate, which are not supposed to be there.

So, I assume, the ccdb gene in my vector has mutated or the TOP10 cells have gone resistant to carbenacillin or ccdb. is there any other possibility ?

it somehow doesnt connect to problem I am facing about getting empty vector after LR reaction.

So, I have done the transformation in BL21 cells which were ok, as there were no colonies on plate with pDest vector.

But my LR reaction hasnt work in them. I still didnt get any colonies.

Atul Pawar on Tue Jan 20 09:21:28 2015 said:

So, I have done the transformation in BL21 cells which were ok, as there were no colonies on plate with pDest vector.

But my LR reaction hasnt work in them. I still didnt get any colonies.

I have not read the entire discussion, but just a question: have you ever had a positive control, to see of the LR reaction itself worked?

The LR reaction mix is very very very sensitive. I only freeze thaw it once! It becomes useless very fast I noticed.

Also, the BL21 strain of E. coli is a very poor choice. It has about 2-3 order of magnitude less tranformation efficiency compared to most cloning strains such as DH10a, DH5a, XL-1, etc. This could be your entire problem (although many other things could also go wrong). I would strongly recommend that you quantify your transformation efficiency. Take a plasmid with the same antibiotic resistance as your target, measure the concentration, then do serial 10x dilutions down to 10 pg/ul. Transform 1 ul of each dilution into your test cells and count colonies. Calculate the colony count per microgram of transformed DNA. Good cells should show 10^8, excellent should be 10^9. Yours will likely be at 10^5.