Point mutagenesis..... - (Feb/19/2013 )
I need to change aminoacid at 2 places in my wild type cloned in pEGFP-N1 vector and also to change aminoacid at 2 places in plasmid which already contains one aminoacid change...I bought reagents from stratagene instead of buying agilent point mutagenesis kit...I tried doing mutations, with 15pmol/microlitre primer concentration with 50ng & 100ng template. But i could not see the pcr products on the gel...I did Dpn1 digestion & transformed too..I got 2-3 colonies but negative plates did not have colonies but it does not contains plasmid...
Please help me out regarding this....
you got to give more specific details about your PCR condition and your proceedings.
The Reaction i set was for 40 microlitre:
Template DNA-10microlitre( 100ng)
Forward Primer-1.5 microlitre (10picomol/microlitre)
Reverse Primer-1.5 microlitre (10picomol/microlitre)
dNTP mix - 2.0 microlitre (0.2mM)
10X Reaction buffer- 4 microlitre
Pfu polymerase- 1 microlitre (2.5U/microlitre)
Double dis.H20- 20 microlitre
This is my pcr condition:
95 degrees- 1min
95 -30sec
68 - 30sec
68 - 7min
In this condition i did both 16 cycles & 25 cycles
Thanks for your kind reply
..As i said before i need to mutate two plasmids, so i went with those plasmids...I have also attached the gel picture which i have done after my PCR in ppt...Look at them too and give your suggestions
The Reaction i set was for 40 microlitre:
Template DNA-10microlitre( 100ng)
Forward Primer-1.5 microlitre (10picomol/microlitre)
Reverse Primer-1.5 microlitre (10picomol/microlitre)
dNTP mix - 2.0 microlitre (0.2mM)
10X Reaction buffer- 4 microlitre
Pfu polymerase- 1 microlitre (2.5U/microlitre)
Double dis.H20- 20 microlitre
This is my pcr condition:
95 degrees- 1min
95 -30sec
68 - 30sec
68 - 7min
In this condition i did both 16 cycles & 25 cycles
AS far the PCR , are you sticking up with the same annealing temp? you probably shd do a gradient starting from 60 degrees and ascend with 2 degree intervals. i believe you designed primers are in accordance with stratagene site direct mutagenesis guidelines. if i am right, the pcr in lane 1 and 3 has got band, is it the right size?? if that s the specific band then i would rather run all the products, and do gel extraction, after which u can do the dpn digestion. usually 1 hr of dpn digestion shd be fine. if that is not the specific one you still got to standandarize the PCR. the catalogue say you might not see the band but i believe you shd see atleast a faint band if you run the whole product otherwise you still got to work on PCR itself. Alternatively you can do without gel extraction provided you get the specific product.
16 cycles is maximum as per me, so try to standardize ur pcr with this cycle itself. and i believe ur plasmid size is around 7kb.
may be now you got to do the gradient and see if you are lucky to visualize the product.
even if your PCR is set, you might still get screwed with the poor efficiency of the competent cells, so you shd have evidence to rule out this is not due to the poor competency of the cells.
good luck.
Thanks for ur reply...Will try and get back....
Hi Gnana...
Hope u remember me... Atlast i got my mutants done..Thank u so much for helping me with all the stuffs..especially top 10 competent cells preparation was very helpful. Thank you so much