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#1 shawncalifornia

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Posted 07 December 2011 - 09:23 PM

Hello
I am using a geneart product (synthesized gene of my interest in a lenti back bone).
They put 5ug lyophilized vector on it, after adding water to it I used 1ul of that for trasformation 200 ul of mgcl2-cacl2 competent cells, but I didnt detect any clony, I checked my competen, my plates, antibiotic anything you think but every thing was ok when I used my previous vectors.
I checked the concentration of the original batch and that was very low, 10ng/ul instead of 100ng/ul.
So I have 2 question what can cause damage to a vector? (I want to understand if I had a mistake in handling or not?).
Do you know a better transformation protocol better than general one (30 min ice, 90 s 42, 45 min 37, then plating on antibiotic plate).?
Thank in advance.

#2 bob1

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Posted 08 December 2011 - 06:31 PM

How did your control plates look?

#3 shawncalifornia

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Posted 08 December 2011 - 11:31 PM

They look good, I saw enough colony in them, it seems it works with my control but not with my original vectors.
By the way the concentration of my control vectors was 1ug/ul and I used 1ul for transformation, it means I used 1ug, while in my samples I used 1ul from a 100ng/ul.
I undrestand it now and I am confused about the large quantity which I have used on my control however there was colony after with this huge concentration transformation??

#4 pDNA

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Posted 09 December 2011 - 01:23 AM

lenti vectors can be tricky! ...and it seems that geneart haven't sent you the 5 µg they have promised (that happens from time to time ...when plasmids are tricky to prep ...then they have a lot of genomic DNA in it ...you should check the DNA they sent you for high molecular DNA that stays in the pockets on an agarose gel).

Another reason for low DNA concentration could be degradation by nucleases ...but i assume you used steril water and nothing else?
Is the DNA conc. you are referring to due to spectrophotometric readings or have you checked the concentration on an agarose gel?

You prepare the chemical competent cells on your own? ...if you spent a lot of money on the synthetic DNA construct i would also spent money on definde, purchased competent cells. With bought chemical competent cells you normally spent pg-ng of DNA ...and not µg! Transformation efficiency is also dependent on using a DNA concentration in a certain range ...since when using to much DNA the efficiency drops.

You can have a look at the protocol NEB suggest for using chemical competent cells ...have a look here.

When working with lentiviral vectors you can also consider working with special E. coli cells that tolerate terminal repeats better than others ...e.g. STBL or SURE cells.

Good luck!

Best regards,
p




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