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Have you monitored your transfection efficiency?
Or you can transfect other easy-transfected cells(293T or other) for confirmating your constructs.
Good luck!
#142168 PDCD4 Plasmid, RAW 264.7 not working Help
Posted
on 24 September 2012 - 04:50 PM
#134659 Site-directed mutagenesis
Posted
on 17 May 2012 - 03:43 AM
Alisa, on 13 May 2012 - 07:49 PM, said:
Hi, I've done mutagenesis for 3 month, but until now all of my colonies are wild type. My vector is pDEST26 (7.3kb) and target gene is 3kb ,so total gene is about 12kb. I've try lots of conditions:
1. Used
10x reation buf. 5ul
DNA template 50ng
f-primer 1ul
r-primer 1ul
dntp 1ul
ddH2O 36ul
pfu 1ul
TOTAL 50ul
95℃-->30sec
55℃-->1min
68℃-->12min (30cycles)
68℃-->5min
DpnI digest for 1hr, add 4ul DpnI digested product to 50 ul competent cell(DH5a)
2ml SOC incubate 6hr
incubate on plate(amp+)
@this condition I have lots of colonies but they are not mutant!
2. Used
So I change my tem to 62℃, again I can't get any mutatant!
3. Use pfu from fermantas and change primer (design from
I can't get any mutatant again!
I am no idea now!!
One of my friends said that I should use
Anyone who has better ideas please tell me!
Thank you!
1. Used
Stragene QuikChange II site directed mutagenesis Kit
10x reation buf. 5ul
DNA template 50ng
f-primer 1ul
r-primer 1ul
dntp 1ul
ddH2O 36ul
pfu 1ul
TOTAL 50ul
95
℃-->1 min95℃-->30sec
55℃-->1min
68℃-->12min (30cycles)
68℃-->5min
DpnI digest for 1hr, add 4ul DpnI digested product to 50 ul competent cell(DH5a)
2ml SOC incubate 6hr
incubate on plate(amp+)
@this condition I have lots of colonies but they are not mutant!
2. Used
Stragene QuikChange II site directed mutagenesis Kit
I asked Stragene. They suggest that I can raise anneal tem. to Tm-3
℃.So I change my tem to 62℃, again I can't get any mutatant!
3. Use pfu from fermantas and change primer (design from
Stragene:
QuikChange Primer Design)
I can't get any mutatant again!
I am no idea now!!
One of my friends said that I should use
Stragene's pfu (for large vectors), it's that true? But it's so expensive!!
Anyone who has better ideas please tell me!
Thank you!
Hi, would you like to upload your primers? And the primer's Tm should be calculated according to the Stratgene's manuscripts.Your insert is about 3kb and your vector is about~7.3kb so why you calculated results is 12kb? If this, you should follow the 3Kb+7.3Kb=10.3Kb and could used the 10min 20seconds for you annealing time. If prolong the annealing time, I think this may affect the Pfu's high fidelity.
What's more, you should make your Tm check, and you could have a gradient Tm PCR to see which Tm is Ok and the best choice is you'd better to rise your Tm to enhence the specificity and the mispairing, I had a case that I used a low Tm to perform my PCR but the mutant is sometimes wrong clone for the primer's binding problems. So the Tm is important to make a sense to your mutagenesis.
Moreover, I want to know whether you have make a control, no primer PCR group and treatment same as your experimental group, If you done this, you may exclude the factor---Dpn I treatment. I usually use this for my trials, this is really important for convincing your results,after all identifying clones must be sequenced.
I have done many mutant, including 1-3 amino acids, deletion,replacement, etc. All use the 18cycles could work efficiently. The average mutagenesis efficiency reach almost 60%(Pick up 3 clones may gain at least 1 mutated clone)
I never purchase this high price strategene mutagenesis kit and just purchase the KOD-plus polymerase and Dpn I. The competent cells used is self-made. And the clones usually grow >100clones. I used this PCR reaction system to perform my mutagenesis: 20-40ng template for 50ul reaction volume(for my trials, I have lowered the reaction volume to 25 ul) and the primer usually~ 1ul sometimes followed the stratgene's manuscripts it may calculated up to 1.1-1.3ul for 50 ul reaction vol.
And 10ul to run the 1% or less concentration AGE for detection your PCR products usually it may never see band in your reaction system without primer. So usually, I could detect the amplification products in my PCR and followed the next Dpn I treatment( Just pipeting up 10-20ul pcr products and add 15-20U Dpn I for treating 1hr in water bath) and then used 1-2 ul transformed into 100ul E.coli JM109. and adding 800-900ul for further culture 45min-60min and spreadering on selective plate with 200ul cultures.
Sometimes the clones may be less but I still gained the clones and sometimes I gained more than 500 clones and randomly pick up 3 clones for sequencing could be enough gained at least correct clones! The clones usually is mutated or un-mutaed without mistakes. Maybe it is so luck for my trials, but I think it won't ony by fortune.
