GroEL from Wolbachia - GroEL from Wolbachia (Jul/01/2010 )

hi people!

i've already posted this problem of mine in this forum.
still awaiting a solution...

am trying to amplify a gene - GroEL from the Endosymbiont Wolbachia of Wuchereria bancrofti.
its a 1.65 kb gene.
i use bangalore genei's PR pol for amplifying my gene cos after amplification i have to proceed with cloning.
i will be using the invitrogen's topo blunt cloning kit for that.

i have designed my primers with gc content 30 and 45% respectively and their Tm 61.2 and 64.3 respectively.
length of primers are 27 and 20 bps respectively.

other reagents and their quantities goes this way:

water - 15.5
buffer - 5.0 (10 X PR buffer banglore genei)
dNTPs - 2.0 (10 mM - finnzyme)
P F' - 1.0 (sigma)
P R' - 1.0
PR pol - 0.5 (banglore genei)
template - 5.0 (from microfilaria - wuchereria bancrofti)

so a total of 30.0 micro liter reaction.

my pcr program goes this way:

95 - 5 mins
95 - 45 secs
58.5 - 1 min
72 - 1 min
40 cycles
72 - 10 mins

i give a good 40 cycles cos approximately 2 months back wen i tried amplifying this gene for the first time, i used to get faint bands only wen the cycles were increased from 35 to 40.
however, am not getting any band now, i either get only un-utilized reagents at the bottom of the lane or its like the entire lane is smeared.

every time i set up a pcr reaction, i isolate fresh dna, means to say the possibility of frequent freezing and thawing can be omitted.

please suggest the necessary measures to be taken.......

thank you people.....

With a 30 ul reaction, you should be using 3 ul of 10x buffer, not 5. I also guess you have too much DNA, both in ug and in ul. Try again diluting your DNA 10x and 100x and adding only 1 ul. Also, Wolbachia has very low GC content, and you could be seeing problems from too high an extension temperature. I would definitely run the extension at 68 instead of 72, possibly as low as 65. I assume you are making up the reactions as a master mix, to reduce variation from inaccurate pipetting. Until you get it working, I would also be doing a 2 minute extension, rather than 1 minute. 1.65 kb probably will work at 1 minute, but it is at the edge.

hello phage!
tanx for the suggestions......!!!

regarding the buffer conc, i told u i used to get a faint band wen i tried amplifying the gene 2 months back....
i never got that faint band even, wen i was using 3 micro liter of 10X buffer.
thats y i started using 5 microliters.

as far as the conc of DNA is concerned, i have tried it with 10X dilution and juz like u said & i used 1, 3 & 5 micro liters of both diluted DNA and also undiluted DNA........ still the same, means, i get either smearing or absolutely no band at all...... smearing wen i use 5 microliters and no band wen i use 3 or 1 microliter....

i hav no idea regarding the extension temperature, the protocol was actually given to me by my senior, who says someone had already tried amplifying this gene (partial fragment) and had optimised it...... so i was asked to stick to this protocol. but may be i shall try ur suggestions.

i dont make master mix, i pipette every reagent for every single reaction i do......

and with the extention time, think i shd try increasing it........

anyways, thank you so much for the timely help....... hope it works out........ and in case if it dosen't can i still contact u for help?? in that case may i have ur mail id please........

regards

S.Prathibha