transformation efficiency - (Aug/25/2010 )

hey all

I have got 2 questions and I would be grateful if any one could help with any of them
1- is there any limitations regarding the respective size of insert and vector, for example is it possible to clone a 7.5 kb fragment in a plasmid of 5.8 kb size?
2- when transforming a bacteria with a plasmid of rather large size, say 13 kb, using cacl2 method, what one can do to increase the efficiency to the maximum level?

thank you all
Mahsa

mahsa on Wed Aug 25 07:01:07 2010 said:

1-The only limit is your ability to process large DNA fragments. It is certainly possible to ligate a 190kb DNA fragment to a 10kb vector.
2-Hmm... I can't help with that. I use electroporation.

Hi Dear,

Surely size is not much limitation in Bacterial transformation.

But increase efficiency of your competent cells with Inoue or Hanhann methods. As max efficiency , You can get with Cacl2 method is 10*6, Which is fairly low for cloning purposes.

Sure i would like to recommend you to prepare competent cells with either of the 2 methods, Keep ligation reactions at 16 degree for 16 hours.

I think things should work.

With Thanks and Regards,

Mole .

perneseblue on Thu Aug 26 04:28:59 2010 said:

tnx a lot. why do you prefer to electroporate them? is it because of their size?

mole on Thu Aug 26 07:43:01 2010 said:

tnx a lot dear mole
U know, I kinda am used to cacl2 method, and , mmmm, and i guess i have to look up the other two. incubating the reaction in 16 degrees increases the efficiency, and another thing is to deactivate the ligase right before in 65 degrees.
have you got any recipes for Inoue or Hanhann, other than Sambrook protocols?

all the luck
Mahsa

Hello Dear,

U can directly use for ligation mix for transformation, No need to heat inactivate DNA Ligase.

Another thing u can use Protocols for Inoue or Hanhann method from Sambrook and Russel (Molecular cloning Volume 01. ).

But keep in mind whenever you are preparing reagent use ultrapure reagents ( Even water).

This will take care of not only contamination But Efficiency also.

Please feel free to ask if something i can answer.

With Thanks and Regards,

Mole.

mahsa on Fri Aug 27 14:48:03 2010 said:

I prefer electroporation because it is fast and has a higher overall efficiency compared to chemical transformation. You can get an efficiency of 10⁸ per ug DNA for even home made electrocompetent cells.

And yes, it is easier to transform large BAC into cells using electroporation.

mahsa on Fri Aug 27 14:57:46 2010 said:

hi, I've been having trouble with cloning recently but I have a few tips for you to increase your efficiency

Use fresh LB for starters! Using old LB (in my case: >2months) severely decreased my transformation efficiency (10-40 (ten to forty) fold)
Prepare 1 liter LB like this:
25g Broth (tryptone 10g, yeast extract 5g, NaCL 10g)
1000ml MQ H2O (Resistance 18,2 MOhm/cm)
Autoclave (in pressure cooker) and store it at 4C in the dark

If you use chemocompetent cells, follow the following protocol for efficiency of 1-5*10^7 (with NEB subcloning efficiency DH5a)
(based on hannahan)

-Add 50ul competent cells to a maximum of 100ng DNA in a maximum volume of 25ul. NO NEED TO MIX! Until after the recovery, be very careful! Dropping the tube 10cm can decrease efficiency 3-fold!!
-Incubate on ice 30 minutes
-Heat shock in heatblock @ 42C 45s
-Incubate on ice 5 min
-Add 500ul LB @RT
-Incubate in shaking incubator 1h @ 37C (recovery step)
-Spin down 1 min @ 7k, remove 520ul (or whatever leaves a convenient enough volume to apply on the plates) medium
-Suspend pellet by vortexing and plate on LB/AGAR plates with appropriate antibiotic
-Incubate O/N @ 37C

Also transform 20pg pUC19 (supplied with the competent cells) or maxiprepped vector as positive control to estimate efficiency

Good luck!