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site directed mutagensis

site directed mutagensis - Protocols and Methods Forum

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  #1  
Old 02-10-2008, 04:25 AM
doaa zineldeen
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Default site directed mutagensis



hello
I just want if anyone have a protocol for pcr mediated site directed
mutagensis without using stratagene kit
i have a template vector containing the insert and i want to mutate k81 into
A i designed 2 primers flanking the mutation site and i want can i perform 2
pcr steps and a third mix with normal polymerase i am using phusion
can i proceed to get the new product containing the mutated sites
If someone can help i am really grateful
Doaa
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  #2  
Old 02-10-2008, 05:10 PM
DK
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Default site directed mutagensis

In article <[Only registered users see links. ].net >, "doaa zineldeen" <[Only registered users see links. ]> wrote:

Below is a protocol I've written for our lab. Note that 1) all Stratagene's
"QuikChange" reactions are NOT PCR but, rather, linear amplification
reactions (the synthesised DNA strands cannot serve as a template
because they are linear), 2) the following protocol is using Stratagen's
"PfuUltra Fusion II" polymerase, which is the same idea as Phusion,
but in our trials was found to work considerably better than Phusion
(perhaps owing to theit high pH reaction buffer).

It is probably more reading than you ever wanted but it gives you
all the background info to be flexible and adjust the protocol to
your particular needs.

************************************************** ***********************************8
Generic QuickChange mutagenesis protocol.
Vadim A. Klenchin, University of Wisconsin, August 2007

Although the regular QuickChange works, it does not always work
well enough. There are several advantages to the new protocol that
I've used now many, many times quite successfully:
- twice less money spent on primers;
- twice less polymerase is used;
- polymerase with lower error rate is used.
Below is a generic protocol followed by notes and comments that
might be helpful.

Reaction mix (25 ul final)
50 ng template
0.2 uM single primer
0.2 mM dNTP mix
2.5 ul 10X PfuUltra II Fusion ("Pfu Fusion" for brevity) buffer
1.0 ul DMSO
water to 24.5 ml
0.5 ul of Pfu Fusion

Cycling conditions
95 3 min
30 cycles:
95 30 sec
55 1 min
65 1 min/kbp template
65 5 min
4 hold

Dpn I treatment and transformation
- Add 1 ml of Dpn I, vortex, spin briefly and incubate for 2 hours
in PCR machine at 37. During incubation, at least once remove
the tube and vortex/spin it again, then continue incubation.
- Electroporate using 2 ml of the reaction, immediately add
0.5 ml SOC, let cells recover for 45 min at 37, plate 200 and
20 ml. The typical result is several-fold less colonies than in
a "standard" QuickChange reaction, and the efficiency in the
50-80% range.


NOTES AND COMMENTS
(in no particular order; I am just mentioning everything that I can
think of being worth consideration)

1. The Pfu Fusion polymerase is a new product that is slightly less
expensive than any of the other polymerases we use.
Its works ~ 4 times faster, makes several-fold less errors than
Pfu Ultra and its reaction buffer has pH value 10.0.

2. The method requires only one primer - just write down the
sequence of the sense strand! It's been found in numerous trials
here that desalted primers from IDT work as well as purified
ones. Save money and time, always order 25 nmol scale desalted
primers.

3. Primer design. Keep primer length between 40 and 60 bp. Try
not to exceed 60 bp because above that size IDT forces you to
order 100 nmol scale with the corresponding price increase.
Ideally, you'd want 15-25 bp on either side of the mismatch, approximately
equal melting temperature (TM) for both "arms" of the primer
(but the "right arm", e.g. 3' part, is more significant because that's
where synthesis starts; try to end it with a couple of G/Cs). The overall
primer TM (not counting the mismatched pairs) should be in the
range of 60-70C (as calculated by IDT's Oligo Analyzer with default
settings). As an example, the most recent primer that worked very
well to delete 6 bases: Left arm 27 bp/51.1, right arm 19 bp/53.4,
overall 46 bp/63.8 and it worked with 55 annealing.

4. In linear amplification applications (sequencing, QuikChange), the
larger number of cycles gives better results. 30X has been tried and
works very well. It is quite possible (but not yet tested) that doing
even more cycles will increase the overall efficiency. You may try
that when doing QuickChange overnight when time is not a
consideration. To be sure, 20X also works, although it was not compared
to 30X side by side.

5. Amount of template. The stated 50 ng is simply lifted from what we
find a near optimum for the standard QuickChanges, 100 ng/50 ul,
and was not tested experimentally. In practice, was 0.5-2 ul minipreps
(depending on culture volume and on plasmid copy number) was used
in the 25 ul reaction. In theory, advantages of higher template
concentration are: i) more product with less number of cycles, ii) higher
chance of priming when annealing is suboptimal. The theoretical
disadvantages of having too much of the template are: i) introducing
more crap into reaction, some of which might be inhibitory (minipreprs are
NOT clean!), ii) increased background if the Dpn I treatment is incomplete,
iii) increase in the misprimed side reactions. There is an optimal balance
between all of these factors somewhere, and it remains to be established.

Other things being equal, the best practice is to have as clean template
as possible and that means: i) don't be greedy with your minipreps; for
high copy number plasmids, 1 ml culture is sufficient, 3 ml is a lot and
more than 3 ml borders on insane; ii) use PB solution volume so that it
covers all areas of the column that were in contact with cell lysate;
iii) use PE solution volume so that it covers all areas of the column
that were in contact with PB, and use PE wash twice.

6. Annealing temperature. Pfu Fusion seems to be less finicky in terms
of annealing conditions. 2-10 degrees below calculated primer TM have
been found to work in various cases. 55 should work in the vast majority
of cases. Note that different program can come up with as much as 15
degrees difference in theoretical TMs! So keep in mind that these values
are somewhat arbitrary.

7. DMSO concentration. DMSO is optional as it rarely makes all or
none difference. Its main effect on the reaction is two-fold: it decreases
melting temperature of DNA duplexes and it reduces primers' secondary
structure. The protocol above uses 4% final. You may also try 0 and 6%
as a substitute to an annealing temperature variance (make sure to
mix DMSO well with other components before adding polymerase).

8. Extension time. Pfu Fusion is quite fast. The default is 60 sec
per kbp at 65C and it definitely works. Tried alternative is 30 sec
at 68C and, for sure, it also works. However, in "QuikChange
cloning" trials we found that 65 gives more colonies
(perhaps because of the some strand displacement activity at 68).

9. Dpn I treatment. This was never optimized at all. Note that
the above protocol uses twice the amount of Dpn I than the
conventional QuickChange (same volume per half the reaction
volume). Whether this is warranted, I don't know. In "QuickChange
cloning", we now use 1 ul DpnI into just 10 ml of QucikChange
reaction in a hope that, perhaps, this increases efficiency of
how it cuts hemi-methylated DNA duplexes. Two hours incubation
is the minimal I tried (same consideration as with the increased
Dpn I amounts). My feeling is that when small volumes are used,
37 incubations in the water bath, incubator, etc are hard to
control and poorly reproducible because of evaporation/condensation,
etc. So do use PCR machine whenever possible (obviously, it is
not an absolute requirement). The vortex/spin thing in the middle of
the incubation is done to increase the exposure of DNA substrate
to the enzyme; I feel it helps, can't see how it can hurt, and it is a
very quick and easy thing to do.

10. Other things that may sometimes be beneficial. Increased
concentration of the primer two-fold, increased concentration of
dNTPs two-fold, an extra 2 mM MgCl2 in the reaction all, in
theory, have an effect of increasing product yield with
concomitant increase in the error rate (not a big deal given the
error rate of Pfu Fusion).

11. Cell recovery after electroporation. I'd recommend doing
it even in the case of ampicillin-resistant plasmids. Feel free to
compare results with and without recovery done on the same
sample and let everyone know of the result. Also, you may try
using LB instead of SOC but this *will* give you
roughly 10 times less colonies.

12. This protocol has now been used to do all kinds of
mutagenesis: single mutations, 33 bases insertions,
2 kpb deletions.

13. Just like regular QuickChange, the single stranded
QuickChange works for two mutations simultaneously but
with reduced efficiency. Make both primers against the same
strand, make sure they have similar TMs and expect 20-30%
of the clones to be double mutants.

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  #3  
Old 02-13-2008, 12:59 AM
Jose de las Heras
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Default site directed mutagensis


"doaa zineldeen" <[Only registered users see links. ]> wrote in message
news:[Only registered users see links. ].n et...

For a point mutation (or a couple) the "Quickchange" PCR system works well.
I know you don't want to use Stratagene's kit... and neither do I. But you
don't need any kit to use the strategy. Read their manual to get a few
hints, and do it with your own reagents... I just sequenced 3 clones out of
8 I picked for a double point mutation (vector+insert size = 6kb)... all
three were perfect. Just one step.

For other things I use a "megaprimer" approach (google) which is another
simple strategy.

Jose


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  #4  
Old 09-08-2009, 07:17 PM
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Default Re: site directed mutagensis

Quote:
Originally Posted by DK View Post
In article <[Only registered users see links. ].net >, "doaa zineldeen" <[Only registered users see links. ]> wrote:

Below is a protocol I've written for our lab. Note that 1) all Stratagene's
"QuikChange" reactions are NOT PCR but, rather, linear amplification
reactions (the synthesised DNA strands cannot serve as a template
because they are linear), 2) the following protocol is using Stratagen's
"PfuUltra Fusion II" polymerase, which is the same idea as Phusion,
but in our trials was found to work considerably better than Phusion
(perhaps owing to theit high pH reaction buffer).

It is probably more reading than you ever wanted but it gives you
all the background info to be flexible and adjust the protocol to
your particular needs.

************************************************** ***********************************8
Generic QuickChange mutagenesis protocol.
Vadim A. Klenchin, University of Wisconsin, August 2007

Although the regular QuickChange works, it does not always work
well enough. There are several advantages to the new protocol that
I've used now many, many times quite successfully:
- twice less money spent on primers;
- twice less polymerase is used;
- polymerase with lower error rate is used.
Below is a generic protocol followed by notes and comments that
might be helpful.

Reaction mix (25 ul final)
50 ng template
0.2 uM single primer
0.2 mM dNTP mix
2.5 ul 10X PfuUltra II Fusion ("Pfu Fusion" for brevity) buffer
1.0 ul DMSO
water to 24.5 ml
0.5 ul of Pfu Fusion

Cycling conditions
95 3 min
30 cycles:
95 30 sec
55 1 min
65 1 min/kbp template
65 5 min
4 hold

Dpn I treatment and transformation
- Add 1 ml of Dpn I, vortex, spin briefly and incubate for 2 hours
in PCR machine at 37. During incubation, at least once remove
the tube and vortex/spin it again, then continue incubation.
- Electroporate using 2 ml of the reaction, immediately add
0.5 ml SOC, let cells recover for 45 min at 37, plate 200 and
20 ml. The typical result is several-fold less colonies than in
a "standard" QuickChange reaction, and the efficiency in the
50-80% range.


NOTES AND COMMENTS
(in no particular order; I am just mentioning everything that I can
think of being worth consideration)

1. The Pfu Fusion polymerase is a new product that is slightly less
expensive than any of the other polymerases we use.
Its works ~ 4 times faster, makes several-fold less errors than
Pfu Ultra and its reaction buffer has pH value 10.0.

2. The method requires only one primer - just write down the
sequence of the sense strand! It's been found in numerous trials
here that desalted primers from IDT work as well as purified
ones. Save money and time, always order 25 nmol scale desalted
primers.

3. Primer design. Keep primer length between 40 and 60 bp. Try
not to exceed 60 bp because above that size IDT forces you to
order 100 nmol scale with the corresponding price increase.
Ideally, you'd want 15-25 bp on either side of the mismatch, approximately
equal melting temperature (TM) for both "arms" of the primer
(but the "right arm", e.g. 3' part, is more significant because that's
where synthesis starts; try to end it with a couple of G/Cs). The overall
primer TM (not counting the mismatched pairs) should be in the
range of 60-70C (as calculated by IDT's Oligo Analyzer with default
settings). As an example, the most recent primer that worked very
well to delete 6 bases: Left arm 27 bp/51.1, right arm 19 bp/53.4,
overall 46 bp/63.8 and it worked with 55 annealing.

4. In linear amplification applications (sequencing, QuikChange), the
larger number of cycles gives better results. 30X has been tried and
works very well. It is quite possible (but not yet tested) that doing
even more cycles will increase the overall efficiency. You may try
that when doing QuickChange overnight when time is not a
consideration. To be sure, 20X also works, although it was not compared
to 30X side by side.

5. Amount of template. The stated 50 ng is simply lifted from what we
find a near optimum for the standard QuickChanges, 100 ng/50 ul,
and was not tested experimentally. In practice, was 0.5-2 ul minipreps
(depending on culture volume and on plasmid copy number) was used
in the 25 ul reaction. In theory, advantages of higher template
concentration are: i) more product with less number of cycles, ii) higher
chance of priming when annealing is suboptimal. The theoretical
disadvantages of having too much of the template are: i) introducing
more crap into reaction, some of which might be inhibitory (minipreprs are
NOT clean!), ii) increased background if the Dpn I treatment is incomplete,
iii) increase in the misprimed side reactions. There is an optimal balance
between all of these factors somewhere, and it remains to be established.

Other things being equal, the best practice is to have as clean template
as possible and that means: i) don't be greedy with your minipreps; for
high copy number plasmids, 1 ml culture is sufficient, 3 ml is a lot and
more than 3 ml borders on insane; ii) use PB solution volume so that it
covers all areas of the column that were in contact with cell lysate;
iii) use PE solution volume so that it covers all areas of the column
that were in contact with PB, and use PE wash twice.

6. Annealing temperature. Pfu Fusion seems to be less finicky in terms
of annealing conditions. 2-10 degrees below calculated primer TM have
been found to work in various cases. 55 should work in the vast majority
of cases. Note that different program can come up with as much as 15
degrees difference in theoretical TMs! So keep in mind that these values
are somewhat arbitrary.

7. DMSO concentration. DMSO is optional as it rarely makes all or
none difference. Its main effect on the reaction is two-fold: it decreases
melting temperature of DNA duplexes and it reduces primers' secondary
structure. The protocol above uses 4% final. You may also try 0 and 6%
as a substitute to an annealing temperature variance (make sure to
mix DMSO well with other components before adding polymerase).

8. Extension time. Pfu Fusion is quite fast. The default is 60 sec
per kbp at 65C and it definitely works. Tried alternative is 30 sec
at 68C and, for sure, it also works. However, in "QuikChange
cloning" trials we found that 65 gives more colonies
(perhaps because of the some strand displacement activity at 68).

9. Dpn I treatment. This was never optimized at all. Note that
the above protocol uses twice the amount of Dpn I than the
conventional QuickChange (same volume per half the reaction
volume). Whether this is warranted, I don't know. In "QuickChange
cloning", we now use 1 ul DpnI into just 10 ml of QucikChange
reaction in a hope that, perhaps, this increases efficiency of
how it cuts hemi-methylated DNA duplexes. Two hours incubation
is the minimal I tried (same consideration as with the increased
Dpn I amounts). My feeling is that when small volumes are used,
37 incubations in the water bath, incubator, etc are hard to
control and poorly reproducible because of evaporation/condensation,
etc. So do use PCR machine whenever possible (obviously, it is
not an absolute requirement). The vortex/spin thing in the middle of
the incubation is done to increase the exposure of DNA substrate
to the enzyme; I feel it helps, can't see how it can hurt, and it is a
very quick and easy thing to do.

10. Other things that may sometimes be beneficial. Increased
concentration of the primer two-fold, increased concentration of
dNTPs two-fold, an extra 2 mM MgCl2 in the reaction all, in
theory, have an effect of increasing product yield with
concomitant increase in the error rate (not a big deal given the
error rate of Pfu Fusion).

11. Cell recovery after electroporation. I'd recommend doing
it even in the case of ampicillin-resistant plasmids. Feel free to
compare results with and without recovery done on the same
sample and let everyone know of the result. Also, you may try
using LB instead of SOC but this *will* give you
roughly 10 times less colonies.

12. This protocol has now been used to do all kinds of
mutagenesis: single mutations, 33 bases insertions,
2 kpb deletions.

13. Just like regular QuickChange, the single stranded
QuickChange works for two mutations simultaneously but
with reduced efficiency. Make both primers against the same
strand, make sure they have similar TMs and expect 20-30%
of the clones to be double mutants.
Hi,
2 Questions:

- Dpn I works in Pfu/Phusion buffer?
- What strain did you used? DH5X works?

Thanks
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  #5  
Old 09-10-2009, 01:45 PM
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Default Re: site directed mutagensis

Quote:
Originally Posted by Arastho View Post
Hi,
2 Questions:

- Dpn I works in Pfu/Phusion buffer?
- What strain did you used? DH5X works?

Thanks
Dpn1 works in Pfu buffer.
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