transformation failure - transformation failure (Mar/08/2007 )

i am doing cloning first time using "instaTA clone PCR cloning KIT"

as i want to sequence TSH mRNA sequence but after gel purification its concentration becomes so low,that why my sir sugessted to clone .

but i failed .there i am telling pocedure ,for guidance that where i am doing mistake.

size of PCR product is 417,and by visual quantification its concentration was 5-6ng/ul.

so in ligation i used 15ul of PCR product .

i leave ligation mixture overnight at 22c.

and after this stored at -20c(i had palced in freezer b coz at that time my bacterial cells(DH5 alpha were not prepared)
can ligation mixture store at -20c?

2ndly i didnt placed LB-ampicillin agar plates in 37c for20 mintues.is it very important step.

3rd i was slightly deviated (just 1 or 2 minutes)from time span which kit has mentioned.5min,5min,and"2 min" ,5min.Are these minutes very much restricted.(and if someone kindly tell me what hapends in these minutes during transformation procedure).

i will be very grate full if some one guides me,i am newer in this technique.greatly needs help.

thankx.

I don't recommend the gel purification because the yield is always very low. You can purifiy the PCR with a column (qiagen kit or centricon) and send to sequencing. If want to make clones (better sequencing results) you can ligate the pcr with out purification or as told before use a column to purify, transform, can use a qiagen kit to get the purify plasmids and then send to sequence.

today again i dont have received T/A cloning results.

but in previous plates which had remained two overnights at 37c have shown just one white small colony.is this a transformed colony? can i further proceed it ..

and what is the time of incubation in hours for blue white colony ( if any one tells me) becoz today i have seen plates after 20 hr.what we call any colony which grow after 2 overnights?

are my DH 5 alpha become week for transformations ?

plez i need some guidance.

if u want clones using PCR products u can use pGEM Teasy vector
u can even use crude PCR products , screening for positive clones will be a bit more work , but u can get there

first as Merlav suggested , use column purification for PCR products
if the concentration is not good , try to make it more concentrated.
make sure of the ligation insert:vector ratio ,, very important!

Then ligation - do the mix and keep at Room temp over night or at 4 degree ( depends on the vector)
the next day start making competent cells , and do transformation , during that time let the ligation mix be at whatever temp it was kept, no need to change it .

make sure of the OD of the DH5 alpha cells when using for making competent cells, if its not correct ,the transformation efficiency gets affected

the time given for the transformation is very important , according to my experience , for example in case of heat shock treatment if u keep more than the recommended time , the efficiency of the transformation is affected. so do follow that

usually colonies after two days wont be the transformed clones.

before u start ur transformation make sure u add Xgal and IPTG to the LB +amp plates
and keep in 37 degree
actually keeping at RT also sometimes works , so thats not so important .

how do u grow the competent cells? each time new batch or is it provided in the kit?

hope some of this helps
all the best
laxmi