5 replies to this topic

[chicken]

hi  i need help for the screen of cdna library. i have kit made library. i have to scan it doing pcr. but very  very little results comming.can some one help me in this situation.


i pick colonies from plate. in 96 well plaate with 20 ul lb media. overnight  shake 30c. next day dilut and heat shocked in h2o. an then pcr with vector specific primers.  

prblm is no pcr result coming.

any guidence most welcom. thanks

[Rsm]

Do you get a PCR product from your empty vector, or no PCR product at all? Which vector do you use for cloning? How about a positive control?

[chicken]

so much thanks minna for ur reply.

i have superscript premade cdna library.of liver cells. genes are inserted in pcmv sport 6 size 4.4 kb. the product from vector is almost 150 bt in all pcr i get near 200bp. kit contain 1.8 kb avarage product size. bt i m merely getting bands of near 300 and 700 band and no other size product.
i start with spreading given stock on lb amp plates. thenpich colonies and mix in 96 well plates well. (in 0 ul terrific broth) then i take 2ul in 20 ul water, heat shocked it. and run pcr.

am i doing any thing wrong.please help

[Rsm]

So the cDNA library is already inserted into pCMV vector? Do you have a known gene in the pCMV vector, that might serve as positive control? Maybe your PCR does not work well, for 1.8kb (or more) you'll need at least 2min extension time. Another possibility might be contamination of your PCR reaction with a different vector.
I would recommend you to pick the colonies directly into the water you'll need for PCR (15ul?), and then into 96well plate with medium. PCR also provides a terrific heat shock, no need to double on that.

Cheers, Minna

[chicken]

         i  still didnt get any results  with pcr.

in the manual instructions. it is stated that "cdna library is stable for 6 months if properly stored at -80.

and this library is received on 2005.

is this  be a problem of not getting any pcr .

bt i get colonies on lb plate and also get growth in terrific broth.and i have also  got just 10 to 12  colonies out of 600 screening.


i cnt understand where is problem if library is expired then y it appear on plates.



some times i just get smear in pcr not even dimers.bt some times get dimers.  and someties i get bands with simple vector (no inserted gene . size almost 300 kb)

i am so much confusd help me

[chicken]

    

any one answer