need guidence for cdna library
Posted 09 June 2009 - 11:09 PM
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
Posted 09 June 2009 - 11:34 PM
Posted 15 June 2009 - 07:59 PM
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
Posted 15 June 2009 - 08:30 PM
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.
Posted 19 July 2009 - 08:59 PM
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