Login

Join for Free!
118911 members


Fake positive colony in lamda RED reconbination, why?

About microscopic forms of life, including Bacteria, Archea, protozoans, algae and fungi. Topics relating to viruses, viroids and prions also belong here.

Moderator: BioTeam

Fake positive colony in lamda RED reconbination, why?

Postby MXH » Sat Apr 03, 2010 1:14 am

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 30°C 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.
During RED recombination, is electroporation necessary?


Regards

Ma
MXH
Garter
Garter
 
Posts: 8
Joined: Sat Apr 03, 2010 1:03 am

Postby JackBean » Mon Apr 05, 2010 5:56 pm

can you put the colonies to another plate (or liquid media), so that they were growing?
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
User avatar
JackBean
Inland Taipan
Inland Taipan
 
Posts: 5690
Joined: Mon Sep 14, 2009 7:12 pm

Re: Fake positive colony in lamda RED reconbination, why?

Postby MXH » Wed Apr 07, 2010 12:10 am

Thank you Dr. Jackbean

I have put these colonies to both kanamycin plate and kanamycine LB liquid medium.
They can growth on these medium. I purified their genome DNA, and cann't amplify anti-kanamycin gene
This confuse me.
MXH
Garter
Garter
 
Posts: 8
Joined: Sat Apr 03, 2010 1:03 am


Postby JackBean » Wed Apr 07, 2010 5:55 am

Can you amplify the gene from some positive control (like your plasmid before insertion) with the same conditions?
Did you try several dilutions of the gDNA?
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
User avatar
JackBean
Inland Taipan
Inland Taipan
 
Posts: 5690
Joined: Mon Sep 14, 2009 7:12 pm

Postby MXH » Thu Apr 08, 2010 3:16 am

Thank you, JackBean

I can amplify the isocitrate lyase gene (1.3kb, like anti-Kanamycin gene)from all positive control with the same conditions.
But I cann't amplify anti-Kanamycin gene from all positive colonies.

I was frustrated.
MXH
Garter
Garter
 
Posts: 8
Joined: Sat Apr 03, 2010 1:03 am

Postby JackBean » Thu Apr 08, 2010 7:51 am

what do you mean by "like anti-kanamycin gene"? Did you amplify the same gene?
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
User avatar
JackBean
Inland Taipan
Inland Taipan
 
Posts: 5690
Joined: Mon Sep 14, 2009 7:12 pm

Postby MXH » Thu Apr 08, 2010 8:06 am

Sorry,
By use these words, "like anti-Kanamycin gene", I mean the size of isocitrate lyase gene is similar to anti-Kanamycin gene
MXH
Garter
Garter
 
Posts: 8
Joined: Sat Apr 03, 2010 1:03 am

Postby JackBean » Thu Apr 08, 2010 8:21 am

the size doesn't matter. The point is, whether your primers work with your PCR conditions
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
User avatar
JackBean
Inland Taipan
Inland Taipan
 
Posts: 5690
Joined: Mon Sep 14, 2009 7:12 pm

Re:

Postby MXH » Thu Apr 08, 2010 9:04 am

JackBean wrote:the size doesn't matter. The point is, whether your primers work with your PCR conditions


Yes, I say this because I do a control experiment:

Same primers, same PCR conditions. The only exception is that the templete of control experiment is PKD46.
MXH
Garter
Garter
 
Posts: 8
Joined: Sat Apr 03, 2010 1:03 am

Postby JackBean » Thu Apr 08, 2010 9:31 am

your primers can anneal two genes?
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
User avatar
JackBean
Inland Taipan
Inland Taipan
 
Posts: 5690
Joined: Mon Sep 14, 2009 7:12 pm

Re:

Postby MXH » Thu Apr 08, 2010 10:32 am

JackBean wrote:your primers can anneal two genes?


Sorry, I didn't exactly express my opinion.
I do two control experiments:
1, Amplify isocitrate lyase (using primer pairs A), to test whether the genome templete is OK.
2, Amplify anti-kanamycin gene using PKD46 as templete (using primer pairs B), to test whether the PCR system is OK.
MXH
Garter
Garter
 
Posts: 8
Joined: Sat Apr 03, 2010 1:03 am

Postby JackBean » Thu Apr 08, 2010 10:41 am

I see. Do you have some positive control for experiment 2 with primers B?
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
User avatar
JackBean
Inland Taipan
Inland Taipan
 
Posts: 5690
Joined: Mon Sep 14, 2009 7:12 pm

Next

Return to Microbiology

Who is online

Users browsing this forum: No registered users and 1 guest