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Site-directed mutagenesis - (Jan/07/2009 )

Hi,
Ive been doing site-directed mutegenesis for a few weeks. But i couldnt get any colonies!!! Can anyone out there please help? THANK YOU SO MUCH.
Im pretty much follow the Stragene QuikChange II site directed mutagenesis Kit protocol which doesnt seem to help

THese are the primers that im using ...

5'GTT TGC CAG TAT TTT NNNNCC TGG CAG GCC

5'GGC CTG CCA GGNNNNAAA ATA CTG GCA AAC

where N is the mutated bases.

This is what i did...
5ul 10x reaction buffer
X ul ( i tried using from 20ng-50ng no result)
1.5ul primer # 1
1.5ul primer #2
1ul DNTP
1 pFu polymerase
Xul of H20

My condition used is
95oC 30 sec
95oC 30sec
55oC 1 minute
68oC 6 minute ( since im having 5.5kb plasmid -using 6 minute extension time to play safe..)

18 cycles..

As per normal..
1ul of Dpn I to digest for 1.5hrs
thaw 50ul XL-1 cells
add 3.5 ul of digested reaction into cells
Incubate on ice for 30 minutes
heat shock for 45 sec @ 42oC
Add 450ul SOC medium
Incubate and shake at 37oC for 1 hr..

then plate on amp/X-gal/IPTG plate

NOTHING Was found.. no colonies...!!!!

WHats wrong? What have gone wrong?

I used SOC Medium on the control.. It seem fine.. low colonies but at least i found colonies..
But for my plasmid nothing was observed!!! Btw, im using pGL3 vector.

CAn someone please help?
Really appreciate it!

THank you!

-galcrazy-

Hello

1. First we need to check if the reaction is working. I normally take 10ul of reaction out before Dpn I digestion and 10 after Dpn I digestion and run it on the gel. If the reaction is working, you will see bands in both the lanes and if its not, you won't see anything in the lane with reaction after Dpn I.

2. If the reaction is working, go down on the amount of reaction product that you use for digestion. salts do lower the transformation efficiency. 3.5 ul according to me is a lot. I generally use 1 ul.

3. I hope you are using the right antibiotic.

If it still doesn't work, try changing the kit, like XL kit or NEB has a different kit (this is when the reaction is not working).

If that also doesn't work, you can take the longer way of overlap PCR.

Hope it helps.

Best
TC



-T C-

First of all you have to check that the PCR worked. Load on a gel your PCR product and also the DNA template, diluted as much as in your PCR product. Then you check on your gel that you have an amplification in your PCR product.

-Missele-

HiHi..

Thank Missele! THank TC!

Thank you for you guys suggestion.

So it means that if i don't even see band before Dpn I digestion - it means my PCR didnt work? ( i do agree though coz ive been doing normal cloning and transformation before this.. i usually get PCR band.. )
Ya, i don't see any PCR product after amplification but their protocol mention [no band is observed before Dpn I digestion, should continue to proceed with transformation.]

Ya, im using the right antibiotic. Ampillicin.

Do you think is there any problem with the primer design?

Do you think is it all right just to get any pFu polymerase ( maybe from fermentas?) and do as per normal? the QuikChange kit price is quite steep.. : (

The NEB kit - is it the phusion kit? the hot start one?

-galcrazy-

Hi

Is the enzyme okay and is working? Chk the kit with some other reaction which has worked for you.

NEB kit is the Phusion kit.

TC



QUOTE (galcrazy @ Jan 8 2009, 04:57 PM)
HiHi..

Thank Missele! THank TC!

Thank you for you guys suggestion.

So it means that if i don't even see band before Dpn I digestion - it means my PCR didnt work? ( i do agree though coz ive been doing normal cloning and transformation before this.. i usually get PCR band.. )
Ya, i don't see any PCR product after amplification but their protocol mention [no band is observed before Dpn I digestion, should continue to proceed with transformation.]

Ya, im using the right antibiotic. Ampillicin.

Do you think is there any problem with the primer design?

Do you think is it all right just to get any pFu polymerase ( maybe from fermentas?) and do as per normal? the QuikChange kit price is quite steep.. : (

The NEB kit - is it the phusion kit? the hot start one?

-T C-

I always checked after PCR and I always get a visible amplification.
I was performing PCR on 50 ng of DNA, with Pfu turbo polymerase in 50 µL. I loaded 5µL of PCR product and 5 ng of DNA, as a control, to see if I could see an amplifaction.
And I see bright bands (sometime a little less bright)
I used the same PCR conditions as you.
hmmm I don't know what is going wrong

-Missele-

"Do you think is it all right just to get any pFu polymerase ( maybe from fermentas?) and do as per normal? the QuikChange kit price is quite steep.. "



It is not necessary to use QuickChange kit always. I have never used that and my mutations work quite fine. I used a general PCR mixture PrimeSTAR HS (Premix)

Somebody in another lab here recently had problems with the mutations. They were using the QuickChange kit. They have tried changing everything except dNTP. Anyway they tried Premix and it worked.

I don't do mutations frequently. So I don't have a lot of experience.

-kj2008-