Problem with plasmids transformation - (Jun/25/2012 )

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

-Don Custom-

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?

-leelee-

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

-Papaver-

leelee on Mon Jun 25 07:05:59 2012 said:

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 ??

-Don Custom-

Don Custom on Mon Jul 2 12:14:34 2012 said:

Which vector did you use?

-Nayeem991-

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)?

-Papaver-