36 replies to this topic

[phage434]

I would check your NZY+ broth by growing an E. coli culture in it.  I would check my plates by growing an amp resistant coli strain on my plates.

[elidamar]

when I developedmymolecule,had somedifficulties inthe visualization of theprocessed material.My problemis addressed throughnew strategies ofprimersize(decreases the amount ofbases).Try loweringto preventloopingof amino acids.good luck

[HOYAJM]

Hello, I have recently ran into problems with SDM. I am introducing 2 mutations into a plasmid construct. The first of the two went smoothly. Here were my conditions.

50 µl PCR reaction

10X pfu reaction buffer    5 µl
10 mM dNTP mix 1.25 µl
Forward mutagenesis primer 2.0 µl
Reverse mutagenesis primer 2.0 µl
dsDNA template (150 ng)    2.0 µl
pfu Ultra fusion II polymerase 1.0 µl
H₂O 36.75 µl

TOTAL    50 µl

Thermocycler profile   

95ᵒC – 4’

95ᵒC – 30 sec
50ᵒC – 1’                  X 25
72ᵒC – 16’

72ᵒC – 7’
4ᵒC - ∞

The Tm of all primers is 62.8C. The GC content of the primers for the successful rxn was 47%.

Now, I used the same profile for the 2nd mutation and got no colonies. The Tm of the primers was 62.8C and the GC content was 57%. The template was the same and all conditions were the same. I think it has to be the annealing temperature. Would the 10% difference in GC content between the reactions cause the conditions not to work for reaction 2? Do you think it would help to raise the annealing temperature to say 55C?

Any input would be greatly appreciated.

Thanks

Edited by HOYAJM, 17 February 2012 - 08:10 AM.

[Jason_LuYZ]

yean_ny_nie, on 09 April 2009 - 04:58 PM, said:

T C, on Apr 9 2009, 01:58 PM, said:

Hey,

You forgot to add the enzyme or forgot to write it ?

Best,
TC

yean_ny_nie, on Apr 9 2009, 09:34 AM, said:

phage434, on Apr 9 2009, 10:52 AM, said:

Your PCR is not working.  Try lowering your annealing temperature.  Make sure your extension time is sufficiently long.  After a day or so, redesign your primers.

Make sure you have sufficient template if you are doing only the recommended number of cycles.  This  is one of the few times when the amount of template can matter in a PCR reaction, since you are trying to avoid over-amplification of the plasmid, and need sufficient template so that few cycles will give sufficient product.

I prepare the sample reaction as below:
1ul of 10 reaction buffer
1ul (25ng) of 25ug/ml dsDNA template
1ul (125ng) of 125ug/ml forward primer
1ul (125ng) of 125ug/ml reverse primer
1ul of dNTP mix
Top up to 50ul with dH2O

The cycling parameter are as below:
i) 95oC for 30secs
ii) 95oC for 30secs
iii) 55oC for 1min
iv) 68oC for 1min/kb of plasmid length (mine would be 6mins)
(ii) to (iv) is for 16 cycles.

I wonder where goes wrong. Please help.  

opps.. sorry.. ya i forgot to write in..  
1 ul (2.5U/ul) of PfuUltra HF DNA polymerase were added to the sample reaction.

I use the KOD-Plus(my old friend used the TAKARA HS STAR Polymerase) instead of using the high price Strategene site-directed mutagenesis kit. I have gained about ~ 19 mutaed clones, and find it is so easy to make a point mutation. The key step is obvious the primer disigning and calculating the Tm. The Tm calculation  used the method refferref in the manuscript in the Strategene kit. I ususally used the 18 cycles to amplify and usually 20-40ng per 50 ul reaction volume and 10 ul PCR products definitely make your to observe the clear band in ~1% AGE detection.

I want to upload a figure to you but I don't know how to paste it in this Bioforum.

From my accumulating experience, I feel the mutagenesis is so easy to indertaking and need all of your reagent in good condition.

The primer: need calculating Tm,well designed and should be synthetised at least with  a minimum purity of PAGE purification grade, the dilution procedure is 100uM stocking solution diluted with TE buffer and dilutied with ddH2O(Millipore) to 10 uM working solution.

PCR is used uniformly followed the enzyme you used and used 18 cycles definitly working very well. I make 3 AA(amino acids)  deletion and mutated to 3 Ala, point mutation and double AA mutation. All are work well. What's more, I just pick up 3 clones for sequencing cloning, the averaging positive clones ration almost more than 60 %.

So, take more cautious to your primer and PCR. But it should be mentioned that the Dpn I treatment is still a critical step. So load less template as possible  is good for your mutagenesis.

Anyhow, Good lucky. If you want to have see my result figure you may sending me a email and disscuss something about this.

Edited by Jason_LuYZ, 16 May 2012 - 01:23 AM.

[fighter]

Hello all,
   I am also trying to mutate a vector. My problem is that I am getting colonies in second day and they are growing very slow in liquid medium. I am wondering is it worth going to isolate DNA and go for sequencing ? i did not use any kit but used biorad iproof HF polymerase and transformed E Coli after DpnI digestion, purification and ligation. If anyone have any suggestions that would be a great help. Thanks.

[Jason_LuYZ]

There are many factors maybe accout for the clonies grow slowly in liquid medium:  the medium is newly prepared or stored for a long time; and the host becterium physiological conditions. Moreover, which E.coli you used in your trials? DH5a usually grow slowly in my experience.
Of course, you should send the clones for sequencing to determine whether your clones is mutant or not.

Good luck!

fighter, on 23 May 2012 - 08:40 PM, said:

Hello all,
   I am also trying to mutate a vector. My problem is that I am getting colonies in second day and they are growing very slow in liquid medium. I am wondering is it worth going to isolate DNA and go for sequencing ? i did not use any kit but used biorad iproof HF polymerase and transformed E Coli after DpnI digestion, purification and ligation. If anyone have any suggestions that would be a great help. Thanks.

Edited by Jason_LuYZ, 24 May 2012 - 09:13 PM.

[fighter]

yes I was using DH5a. But this is something to do with the mutated construct because if I transform original vector, I am getting enough colonies by overnight. Transformation after mutation gives colonies after 48 hours. Any way I will isolate DNA and check. Thanks for your reply.