Help me out with Primer calculation for point mutagenesis.... - (Feb/21/2013 )

Can u guys plz help me out in preparing stock & working of primers for doing point mutations....

Herewith i have attached the primer details for your suggestions...

Thanks in advance... If possible also suggests me with the annealing temperature...

you will get 100 micro molar final conc of each primers if you dissolve in the appropriate volume they have mentioned in volume-pmol/microlitre. this will be your stock.

but how i usually do is for mutagenesis i use 125 ng of primers in total. so i would suggest you to dissolve as they mentioned but calculate like below,

for Primer 1 in ur sheet:
594 microlitres will give you 100micromolar final concentration this is one way.... and the other is ,they also mentioned it is 805 micrograms so now if you dissolve 805 mcirograms in 594 volume (just not to alter the universal primer stock conc_100miromolar) this will give you-1.36 micrograms/microlitre (805/594), so now your stock is 1.36 microgram/microlitre or 100micromolar.

to get 100ng/microlitre (working) - 0.1/1.36x100 (total volume) = 7.35

so to prepare 100 ng/microlitre - 7.35 (stock primer)+ 92.65mcirolitres (H2O).

now you can use 1.25 microlitres of each primers per pcr to get 125ng of each primer for a reaction.

good luck....

note: i would go with 63 or 65 as annealing temp for this primer....try out....

For that primer i went for 68 &55 as annealing temp but that did not work ..I will try now with 63 & 65...
Thank u so much

HI GNANA....
I tried as per ur suggestions...for that 284D primer & for 353 D primer @ 63 & 65... I got some faint band but i dont know whether its my template (100ng) which i have used for the reaction...Give ur suggestions by seeing the picture...so that i may proceed with Dpn1..If u suggest me to go to Restriction...Also give idea abt the reaction i need to follow...

Will be waiting for your reply

Sorry i forgot to attach my picture & the reaction details...help me out

kartiga natarajan on Mon Feb 25 14:00:54 2013 said:

when i see comapring the 63 the 65 degree PCR looks like having a faint PCR band...fine, just proceed with Dpn digestion, i dnt know what Dpn you are using, the kit provided? if KITs jus follow as they suggested for Dpn digestion or NEB enzyme? i use NEBs Dpn, which i directly add 1 microlitre or slight over 1 microlitre in the PCR, incubate an hour at 37 degrees then i do purify and proceed for transformation. but purification is just optional. make sure you have good competent cells.

Me too using Dpn1 from NEB...So Is this reaction ok?

Reaction for 50 microlitre
Pcr Product- 40microlitre
10X NEB buffer4- 5 microlitre
Dpn1-------------1 microlitre
H20----------------4 microlitre

Incubate at 37 for 1 hour...Is this ok? Thanks in advance for your helping hand

ya sounds fine, but since you added 100ng plasmid i would use like 1.2 Dpn for an hour....otherwise i am ok with urs.

good luck...