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pkD46 vector growth in E.coli problem

Gene Knockout E.coli

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8 replies to this topic

#1 Inbox

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Posted 02 August 2012 - 04:07 AM

I tried to transform a pkD46 in DH5 alpha strain an grow in LB - amp plate but did not get colony even after 24hrs. Any suggesting welcome.

#2 leelee

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Posted 02 August 2012 - 06:13 AM

Can you give us some more info, so we can help? Such as:

- were you give the plasmid, or did you purify it yourself? If so, how? Did you quantify it? Check it on a gel?

- how did you make your DH5a competent? Did you do a transformation control (to see if your cells are in fact competent)?

- how did you transform the bacteria? What was your protocol?

- what concentration of amp did you have in you plates? What temperature did you put your plate at? (pKD46 is temperature sensitive)

#3 Inbox

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Posted 02 August 2012 - 09:27 PM

Hey thanks for reply.
I was given plasmid, I did not purify it myself. I transformed 40 ng plasmid (Nanodrop reading). I did not check it on gel. I am using TSS protocol for making cell competent. I do used positive control with PUC18 to check whether cells are competent (30 degree incubation). I am using following trasformation protocol
1. thaw competent cell for 30 min.,in ice
2. adding 40ng desired plasmid,
3. putting in ice for 20 min,
4. heat shock 42 degreee 1min. 30 sec.
5. keep in ice for 20 min.
6. add LB. 1 ml
7. keep in incubator 30 degree
8. centrifuge at 4000 rpm. 5 min.
9. remove supernatant by inverting tube quickly.
10. mix remaining approx. 100 microlit supernatant with pellet.
11. plate on LB- amp plate [100 microit of ampicilin (50mg per ml stock) ]
12. incubate at 30 degree for 24 hrs.


I know pkd 46 is temperature sensitive, so growing it at 30 degree.
I thought low copy number plasmid will require more time. Now its 48hr incubation still no colony found.

#4 leelee

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Posted 02 August 2012 - 10:37 PM

When you say 100 ul of amp- in how many ml of LB agar is this? What is your final concentration?

#5 Inbox

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Posted 03 August 2012 - 06:07 AM

100 ul of amp- in 100 ml of LB agar. Final concentration of 50 micro-gram/ ml of LB.

#6 pDNA

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Posted 05 August 2012 - 12:15 PM

pKD46 is a somehow tricky vector ...i regularily exprienced problems with it. It tends to be unstable in certain host backgrounds ...maybe due to basal expression of the araB promoter.
An alternative is to use the pSIM plasmids from the Court lab.

Regards,
p

#7 Inbox

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Posted 06 August 2012 - 10:15 PM

I am using DH 5 alpha cells for transforming it. Can you please suggest cells suitable for its transformation. What is minimal concentration of vector essential for transformation.

Thanks.

#8 klenow

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Posted 07 August 2012 - 02:29 AM

First I would run a gel to check the integrity and the proper size of the vector (you can run undigested vector and also run digested vector and check that the bands have the expected size). The vector might be degradated or being a wrong vector... If this is not the case, then you could try using electrocompetent cells and check if you obtain positive colonies...If not, you might also try to modify your chemiocompetent protocol: heat-shock time: 30-45 sec is working for me. Although a heat-block can be used, if you have problems you may try to use a water bath for the heat shock...
For my experience, Amp resistant vector can be plate directly onto LB agar plates+Amp without growing them.
Try to use a positive control (ask some college to give you another ampR plasmid) , you mention something but it is not clear if is working or not...
Good luck

#9 pDNA

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Posted 07 August 2012 - 06:04 AM

when we used pKD46 we did all the knock-outs or knock-ins in MG1655 and then moved the traits using P1 transduction in the strain of choice.

Regards,
p





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