Hello everyone,I have been trying rescue cloning experiments as per the protocol suggested by http://www.epibio.com/docs/default-source/protocols/ez-tn5-lt-r6k%CE%B3ori-kan-2-gt-tnp-transposome-kit.pdf?sfvrsn=8
but inspite of trying multiple times I am not able to get any colony on the plate.
Can anyone help me with this ?
Hi Abhishek,
What bacteria are you mutagenising?
I Did this for my 2007 MPMI paper (Oxalate-Degrading Bacteria Can Protect Arabidopsis thaliana and Crop Plants Against Botrytis cinerea Molecular Plant-Microbe Interactions 12/2007; 20(12):1535-44. DOI:10.1094/MPMI-20-12-1535)·
It also did not work right away, you really need good electromax PIR Ecoli (so epicentre can sell you something else). THe bit that did the trick for me was to do the digestion of gDNA from your mutants in a laerge volume and then precipitate it before ligation:
20 mul DNA in 200 mul restriction mix 3 mul enzyme (I tried Kpn1 and EcoR1), 3 hrs 37 degrees,
Add extra 3 mul enzyme, o/n 37 degrees
Precipitate with NaAc/EtOH and resuspend in 20 mul water.
Self-Ligation: microliters
gDNA 12
lig buffer 10X 1.5
10 muM ATP 0.5
T4 ligase 1
Vend 15
EcoRI ligation, Electroporation into electromax PIR Ecolis
Spread 2X 100 mul on LB with 50 ppm Kanamycin
Most lines > 10 colonies,
It may help to first plate them one a lower concentration of Kanamycin and then repick them to >=50 ppm kan.
THese gave low yields in plasmid preps, I needed to do a column midiprep before sequencing.
Good luck with yours!
Hello Henk-Jan
I am working with M.smegmatis MC2155 strain.
I have already got TransforMax™ pir-116 Electrocompetent E. coli for my work.
Hi Abhisek
For me doing the digestion in a large volume and then precipitate it before ligation helped. MAke sure your genomic DNA is fully digested before ligation. Also use maybe more than one enzyme (seperately), it may be that you are unlucky and the sites of your enzyme were far from the integration site, hence making a religated plasmid to big to clone.
THere may be sequences in there that affect E coli, so be patient. you can lso plate your electroporation makes on various plates, spread it over more plates and put some of them at 28 degrees rather than 37 and give them more time!. Some of my rescued colonies were very slow and yielded little plasmidDNA! >24 h at 37 is
When you plate some on lower kan, say +/-20 ppm, repick on to 50. It is better to elminate some false positives by repicking rather than to have nothing at all.....#
Hi Abhishek,
Did you get it to work? I am using the transposome kit and the pir-116 cells now. I had no luck so far, but the control DNA works in the cells though. Do you have any recommendations?
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Methods
- Cloning