6 replies to this topic

[Don Custom]

Hi everyone

I have a problem when i tried to clone 1.7 kb gene into a vector. After transformation using JM109, a lot of colonies grow well on LB+Amp plate, then i selected some of them and inoculated into LB broth+amp for overnight growth. I tried to extract the plasmid from the cultures using homemade alkali lysis reagents. The problem is that when i check the products by running agarose gel electrophoresis there was no band appear at any size (it's totally blank).

First i thought that it would be related to the lower conc. of amp or my  LB+Amp plates were expired but i did spread the stock JM109 without plasmid and there was no colony. That's mean my LB+Amp plates still work and my stock JM109 is not contaminated. I did try with different batch of JM109 but nothing has changed, there were still a lot of colonies on LB plate, the cells grow well in overnight culture but no plasmids found.

Would you please help or give me some suggestions??? Thanks in advance

[leelee]

My guess would be that the problem lies with your plasmid isolation rather than your transformation. How experienced are you at the plasmid isolation procedure? Do you have a kit you could use (even just once) to check if it is your extraction that is failing?

Can you give us your step by step protocol?

[Papaver]

You can additionally run a colony check PCR for your gene to confirm the transformation.

[Don Custom]

leelee, on 24 June 2012 - 11:05 PM, said:

My guess would be that the problem lies with your plasmid isolation rather than your transformation. How experienced are you at the plasmid isolation procedure? Do you have a kit you could use (even just once) to check if it is your extraction that is failing?

Can you give us your step by step protocol?

This is the protocol that i use,

- Transfer 2 ml culture to a tube, centrifuge
- Add 100 ul of resuspension solution, resuspend pellets by pipetting
- Add 200 ul of solution II (200mM NaOH, 1%SDS), mix by inverting
- Stand for 2-3 min, add 200 ul of neutralizing solution (3.0M Potassium Acetate, pH5.5 by glacial acetic acid),mix by inverting, centrifuge
- Remove the supernatant to a new tube, add abs. ethanol 900 ul, invert, centrifuge
- Remove sup. , add 500 uL of 70% ethanol, pipetting, vortex, and centrifuge
- Remove sup., air dry the pellets for 30 min at RT
- Resuspend with sterile dH20 30ul and take 5 ul for electrophoresis.

I worked with this protocol in past few months and it didn't has a problem like this time. BTW, the reagents might be in trouble so i'll try with a kit.

[Don Custom]

Dear all,

I did try with two others plasmid extraction kit (QAIGEN and Geneaid) and that didn't change anything, no plasmid has found in any colony.
Also for the PCR for the gene

I tried with a recently prepared LB Agar and also using the commercial competent cells but there're still a lot of colonies grow on the plate and quiet hard to select for a single colony. Is it possible that the colonies that i pick are sattlelite colonies that cover all of the plasmid containing cells ??

[Nayeem991]

Don Custom, on 02 July 2012 - 04:14 AM, said:

Dear all,

I did try with two others plasmid extraction kit (QAIGEN and Geneaid) and that didn't change anything, no plasmid has found in any colony.
Also for the PCR for the gene

I tried with a recently prepared LB Agar and also using the commercial competent cells but there're still a lot of colonies grow on the plate and quiet hard to select for a single colony. Is it possible that the colonies that i pick are sattlelite colonies that cover all of the plasmid containing cells ??

Which vector did you use?

[Papaver]

That's strange....how did you transform the vector (complete protocol including how you plate out the cells). I guess you have checked the vector before you transformed it...can you put a picture of the plates?
Do you plate some controlls, like strain without a plasmid, strain with another plasmid (coding for Amp-resistence)?