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cempetent e.coli and transformation


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#1 mahsa

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Posted 20 December 2009 - 05:52 AM

i tried to prepare HB101 competent cells and transfer them with a plasmid of about 11 kb. i used sambrook's protocol for transformation and the protocol below for preparing competent bacteria and i didn't get any transformed bacteria. i am pretty new in the field and i would appreciate any comment or solution for this matter.
1- add 200 micro liter of bacteria to 40 ml of LB broth and incubate in 37C along with 200 rpm shaking till it reaches the OD600 0.4
2- add 750 micro liter of CaCl2 0.1 molar in a falcon tube and add distilled water to the total volume of 15 mili liter and place it on ice to get it ice-coled.
3- after LB-Bacteria reaches to the OD 0.4 divide it to 2 15 ml falcon tubes and place it on ice for 15 mins
4- then centrifuge them in 4C for 15 min with 5000 rpm
5- discard the supernatant
6- add 3ml of CaCl2 0.1 M ice cold on the pellet and mix it gently then add the remain of 15 ml of CaCl2 and place them on ice for 30 min
7- centrifuge them in 4C for 15 min with 6000 rpm
8- discard the supernatant
9- collect the bacterial competent cells with 1 ml of CaCl2 0.1 M
10- keep the competent cells on ice for 16 hours for they will have a better competency after this period of time


:rolleyes: pleas help meeeeeeeeeee

#2 pDNA

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Posted 20 December 2009 - 08:33 AM

maybe you can use electroporation instead of the CaCl-method ...people often experience problems with large vectors.

Concerning your protocol ...there seems to be no mistake in it!

Beside the size of your vector, another issue could be the integrity of your plasmid ...large plasmids often get degraded by isolation ...check it on an agarose gel.

Regards,
p

#3 molgen

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Posted 21 December 2009 - 12:09 AM

Using RPM isn't a good idea.
Different rotors in the same RPM will give different RCF or g.
You might be using to much force or not enough force.




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