a*****v
发帖数: 128 | 1 Who made such mutations before? Is that possible to make four base mutations
at same time?
Here is my mutated part, CagGcGA (the capital letters are mutated), on both
side I have already added about 15 bp and the Tm is calculated at 78. But I
did not get any PCR products.
Thank you in advance | o*****r
发帖数: 156 | 2 I suspect your primer is too short and the way you calculate Tm is not right
.
go to their website and use the online primer design tool.
I used to mutate 4-6 bases, and always worked (the primers are usually 40-50
bp).
mutations
both
I
【在 a*****v 的大作中提到】
: Who made such mutations before? Is that possible to make four base mutations
: at same time?
: Here is my mutated part, CagGcGA (the capital letters are mutated), on both
: side I have already added about 15 bp and the Tm is calculated at 78. But I
: did not get any PCR products.
: Thank you in advance
| n********k
发帖数: 2818 | 3 shall be an easy job---I would use fusion PCR---less sequencing and always
works...order two overlapping primers with the mutations in the middle, just
make sure there are at least 16-20bp on the 3 primer side. The overlapping
between the two primers (for fusion) should be at least 16-20bp too. Your
primer should be about 6-12bp+CagGcGA+18-20bp about 30-40bp for both primers
. try to keep the amplicons short 100-500bp. Longer is ok but need more
sequencing and also could get more challenging....Forget about those Tm or
whatever rules for plasmid templates. As long as there is no fundamental
errors, pretty much anything would work for plasmid template PCR. By fusion
PCR, one could mutate many different places in a single reaction or a
couple of sequential fusion PCR reactions
In theory, with plasmid template, a primer of12bp-16bp is enough to do PCR
but that would need a good/experience hand.
mutations
both
I
【在 a*****v 的大作中提到】
: Who made such mutations before? Is that possible to make four base mutations
: at same time?
: Here is my mutated part, CagGcGA (the capital letters are mutated), on both
: side I have already added about 15 bp and the Tm is calculated at 78. But I
: did not get any PCR products.
: Thank you in advance
| a*****v
发帖数: 128 | 4 Now I am also thinking about that,
The old primer is only 35bp, that could be the problem,
I will try to order a new one.
thanks！！
right
50
【在 o*****r 的大作中提到】
: I suspect your primer is too short and the way you calculate Tm is not right
: .
: go to their website and use the online primer design tool.
: I used to mutate 4-6 bases, and always worked (the primers are usually 40-50
: bp).
:
: mutations
: both
: I
| a*****v
发帖数: 128 | 5 That is really a Classical way,
But I am not such an experienced hand on that,
If there is not so urgent job, I will try it,
Thank you!!!
just
overlapping
primers
fusion
【在 n********k 的大作中提到】
: shall be an easy job---I would use fusion PCR---less sequencing and always
: works...order two overlapping primers with the mutations in the middle, just
: make sure there are at least 16-20bp on the 3 primer side. The overlapping
: between the two primers (for fusion) should be at least 16-20bp too. Your
: primer should be about 6-12bp+CagGcGA+18-20bp about 30-40bp for both primers
: . try to keep the amplicons short 100-500bp. Longer is ok but need more
: sequencing and also could get more challenging....Forget about those Tm or
: whatever rules for plasmid templates. As long as there is no fundamental
: errors, pretty much anything would work for plasmid template PCR. By fusion
: PCR, one could mutate many different places in a single reaction or a
|
|
