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Fake positive colony in lamda RED reconbination, why? - (Apr/06/2010 )

Hi all,

Recently, I perform a number of pflB-focA gene knock-out using lamda RED recombination in Escherichia coli strain W1485.
But I have a hard problem.
Below is my experiment:
1. PCR amplify linear fragment from both pKD3 and pKD4
Purify PCR product by gel.
2. Make target W1485strain maintaining pKD46 by growing at 30C with 10 mM L-arabinose (E. coli was induced for just 1 h before harvesting)
3. Transform E. coli using Calcium Chlorid.
4. Plate the transformation culture on LB plates supplemented with chloramphenicol (8ug/ml) or kanamycin(40ug/ml).

Here is the results:
1. I can get several dozen colonies from plate with kanamycin, but cann't get any colony from chloramphenicol.
2. I cann't amplify anti-kanamycin gene from these anti-kanamycin strain.
Can anyone tell me what could be the problem.


Regards

Ma

-MXH-

Dear MXH,

first of all ...have you digested your PCR product with DpnI befor or after gel purification to minimize background from your plasmid template? most of the time gel purification do not suffice ...DpnI digest is highly recommended since it cuts the methylated plasmid template only. This should lower false positive clones that contain the plasmid instead of the chromosomal integration.

Additionally, i would recommend to do a control experiment with cells cultured without arabinose ...if you get more colonies with those cells compared to the arabinose treated something went wrong and the colonies you get are not likely to be the product of recombination.

I do not know much about your strain W1485 ...is it recA positive or negative?

Test the viability of your cells after making them competent ...often the viability count drops drastically.

Another tip ...restreak the colonies from the original plate to a new plate containing the corresponding antibiotic ...most of the time false positive colonies aren't able to grow and PCR screening is more reliable on fresh plates because there is no background from the original DNA used for the transformation.

Hope this will help you!

Regards,
p

-pDNA-

Dear pDNA

Thank you very much for your help.


1) :) I didn't digest PCR product with DpnI. I will try it.
But an strange question is that I can get several dozen colonies from plate with kanamycin (perform PCR using PKD4 as templete), but cann't get any colony from chloramphenicol plate (do PCR using PKD3 as templete).
2) :( :lol: I know control experiments is important, but I have not did it before. From now on, I will do a control experiment.
3) W1485 (genotype F+ λ-) strain is U V light treated E.coli K-12 wildtype (F+ λ+).
4) Take knock out pflB gene using anti-kanamicin gene as an example, I have moved colonies from the original plate to two new plates. The first plate contain the kanamycin, the second plate contain kanamycin and ampicillin. Alomost all colonies can growth on new kanamycin plate, several cant growth on plate containing kanamycin and ampicillin.

Regards

Ma

pDNA on Apr 6 2010, 09:30 PM said:

Dear MXH,

first of all ...have you digested your PCR product with DpnI befor or after gel purification to minimize background from your plasmid template? most of the time gel purification do not suffice ...DpnI digest is highly recommended since it cuts the methylated plasmid template only. This should lower false positive clones that contain the plasmid instead of the chromosomal integration.

Additionally, i would recommend to do a control experiment with cells cultured without arabinose ...if you get more colonies with those cells compared to the arabinose treated something went wrong and the colonies you get are not likely to be the product of recombination.

I do not know much about your strain W1485 ...is it recA positive or negative?

Test the viability of your cells after making them competent ...often the viability count drops drastically.

Another tip ...restreak the colonies from the original plate to a new plate containing the corresponding antibiotic ...most of the time false positive colonies aren't able to grow and PCR screening is more reliable on fresh plates because there is no background from the original DNA used for the transformation.

Hope this will help you!

Regards,
p

-MXH-