Hello.
I am trying to clone Jag1 into pGBKT7 vector, but for some reason it does not work and we are very puzzled by our results. Below is everything I did from start to finish:
Jag 1 containing plasmid: pENTR1A-HA-Jag1. backbone size: 2700bp Insert size: 3787bp
pGBKT7 vector: 7.3Kb
1: Plasmid preparation: I culturedpENTR1A-HA-Jag1 and pGBKT7 carrying bacteria with appropriate antibiotics overnight. I pelleted the bacteria and used QIAGEN miniprep reagents P1 P2 and P3 to lyse the cells and release the plasmid. I then performed isopropanol precipitation, which yielded a good amount of DNA pellet. I resuspended the pellet in 20uL endotoxin free TE buffer and used this for restriction enzyme digestion
2: Digestion: I digested pENTR1A-HA-Jag1 with EcorI (5' site) and XhoI (3' site) and pGBKT7 with EcorI (5' site) and SalI HF (3' site). Protocol for 45 uL reaction: 20uL DNA, 1.5 uL Enzyme 1, 1.5 uL enzyme 2, 4.5 uL buffer. incubate at 37 for 1 hour. I ran the product on 1% gel. see picture 1. the brightness is increased and for that reason the bands look very bright, however, in reality they were normal.
3: Gel Purification: I cut out the band of appropriate size, trimmed of as much gel as possible and purified the DNA according to QIAGEN gel extraction kit. I followed the protocol precisely. I measured the DNA concentration of the result using nanodrop. insert concentration: 97.3 ng/uL. Vector conc: 203.6 ng/uL. in both cases 260/230 ratio was very low ~0.2-0.3.
4: ligation: I used NEB T4 DNA ligation kit, 3:1 insert:vector ratio. I ran three trials in which total DNA concentration was 26ug/mL,13.6ug/mL and 8.45 ug/mL. I ligated at room temperature for two hours and heat inactivated at 65C for 10 minutes.
5: Transformation: I thawed commercial competent cells on ice, added 5uL of my ligation, gently pipetted up and down and incubated on ice for 30 minutes. heat shocked at 42C for 30 seconds, placed on ice for 5 minutes, added warm LB and shook for one hour. I plated the cells on LB agar plates with appropriate antibiotic (1:2000 dilution). I also did + and - controls. next day I did not have any colonies on any plate, but on the second day I had normal looking colonies. I had much more colonies on plates which i transformed with 26ug/mL DNA than 8.45ug/mL. negative control did not have any colonies. I picked them using a pipet, dropped in LB broth with antibiotic (1:1000 dilution) and shook overnight.
6: Final digestion: I pelleted the cells and the pellet was orange (see picture 2. left one is transformed bacteria, right one is a some other plasmid containing bacteria). I precipitated DNA using the same method as described, and digested with EcorI and XhoI using the same protocol. I ran on the gel which is shown on picture 3. on the left I have digested DNA and on the right undigested. there is no plasmid for some reason. I don't know how this is possible. Why did the cells grow if there is no plasmid in them.
Thank you very much for taking the time to read this. I really appreciate all the feedback!
Looking through what you have written, I would say the most probable answer is that the colonies you picked from your agar plates were not actually transformants, as it is unlikely E. coli would have taken 2 days to grow up (unless whatever was contained in your plasmid caused some sort of growth defect in E. coli which is unlikely). I would guess that those colonies are probably a contaminating bacteria of some kind, due to the difference of colour. Either that or the antibiotic selection has been unsuccessful and your E. coli survived, and this is why they have taken 2 days to grow. Inefficient antibiotic selection often manifests itself as slow growth. Perhaps your stocks are old, I would remake them or use higher concentrations. However, is is puzzling that you got no cell growth on your negative control.
If those colonies you picked were true transformants and not a contamination, you should first check the concentration of the DNA you recovered by nano drop. You may actually have DNA but its concentration may be quite low. It is also strange that you seem to have quite a lot of DNA stuck up in your gel wells, but I guess this could be chromosomal DNA if you are just harvesting DNA by isopropanol precipitation.
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