I have previously performed knockouts in E.coli using lambda red recombinase many many times. Now I can't do it anymore and I don't know what I'm doing wrong. Please help me!
My standard protocol that previously worked at least 50 times.
0. Use the Keio published primers to amplify a kan/linear fragment from pKD13.
1. Transform a strain with pKD46.
2. Pick a colony or glycerol stock and grow in LB overnight with carbenicillin.
3. Next day - Inoculate 1% into LB with carbenicillin and 1mM L-arabinose (I tried making fresh arabinose already).
4. When OD is around 0.6, put on Ice.
5. Spin down at 4C and wash in 10% glycerol 3 times.
6. Electroporate
7. Put in 30C for 2 hours, or 37C for 1 hour.
8. Plate half on Kan at 37C.
9. If no colonies grow, plate the other half the next day.
What could happen that makes this procedure all of a sudden not work? I have tried different strains, both with freshly prepped pKD46. I also tried using an old glycerol stock strain containing pKD46 that I have used many times. I used to get 50 colonies with 100% efficiency. Now I can't get colonies.
Yo Sammy!
Are you using a strain that already has several deletions? Subsequent deletions can become very complicated.
Its good that you tried other pKD46 harboring cells. Try making all media/solutions fresh. Are you using a new batch of plates? Wrong antibiotic by mistake? Derrick touched something?
Sammy,
Did you find out what was wrong? I am facing similar problem recently.
Hey everyone, I solved my problem. I don't know why, but I was storing kanamycin in the refrigerator. When I stopped this, and stored it in the freezer, my knockouts started working again.
No idea why, but it works.
Hi Sammy! What solvent did you use to make your kanamycin stock? I am guessing you had used an organic solvent that makes you to be able to store it in the freezer. I have also been storing my water-made kanamycin stock in the refrigerator for a long time, and have been having a similar problem with LRR mutant construction. Any useful information would be gladly appreciated.
Hi Olakunle,
I hope your problem is solved by now. If not, maybe try the following
1. The Kanamycin stock can be stored at -20 degrees if it's made in water, and works efficiently easily for upto 2 weeks. Also, use a lower concentration of Kan (25ug/ mL) for the knockouts, as there is only one copy of the gene on the genome.
2. Also, try incubating for longer time after electroporation (~4 hrs) at least, and plate only half of the cells, incubate the rest at 37 degrees.
3. I make 200 uL electrocompetent cells from 20 mL culture, and use 100 uL each for a control (water) and desired DNA (100ng) for electroporation, as sometimes if the cell number is not higher, the recombinase might not be expressed in enough amounts.
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