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Site-directed mutagenesis

mutagenesis DpnI Pfu

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#1 Alisa

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Posted 13 May 2012 - 07:49 PM

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

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 min

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

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!

#2 kaveh

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Posted 13 May 2012 - 08:45 PM

I needed to do a SDM on 12K vector a while back. I didn't have the Stragene kit and decided to give our high fidelity polymerase (AccuPrime Pfu, Invitrogen) a try. I designed the primers with the Strategen web tool and ordered the primers. Since the primers were long, I asked the company to purify them on PAGE (this takes a longer time and more money, so it's not going to be a 10$ per pair primer). Then I borrowed the dpnI from the neighbors and that did it! all my colonies were mutant. Here is my thoughts:

1) Are you sure about your primer design? They have to be overlapping and long.
2) Did you purify the primers?
3) Do you give the polymerase enough time? (12 minutes should be fine, but I would give it a little longer)
4) I might be wrong, but a longer dpnI treatment might help to eliminate more of the original plasmid.
5) How do you check for the mutation? sequencing?

#3 Alisa

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Posted 13 May 2012 - 09:27 PM

I needed to do a SDM on 12K vector a while back. I didn't have the Stragene kit and decided to give our high fidelity polymerase (AccuPrime Pfu, Invitrogen) a try. I designed the primers with the Strategen web tool and ordered the primers. Since the primers were long, I asked the company to purify them on PAGE (this takes a longer time and more money, so it's not going to be a 10$ per pair primer). Then I borrowed the dpnI from the neighbors and that did it! all my colonies were mutant. Here is my thoughts:

1) Are you sure about your primer design? They have to be overlapping and long.
2) Did you purify the primers?
3) Do you give the polymerase enough time? (12 minutes should be fine, but I would give it a little longer)
4) I might be wrong, but a longer dpnI treatment might help to eliminate more of the original plasmid.
5) How do you check for the mutation? sequencing?

Hi
(1)My primer design is from Strategen web tool like you, and my primer is 35bp.
(2) I didn't ask company to purify my primer.
(3) I sent my colonies to sequencing
I'll try longer extension time and DpnI treatment.
Thank you for your advises!

#4 GNANA

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Posted 14 May 2012 - 01:05 AM

i have done lot of SDMs and i never had failure untill now...your protocol looks ok, but your PCR cycle shdnt be 30. may be 15 shd be more than enough. after dpn digestion do a gel purification and transform. my usual primer design tool is Primer X.....i use Accuprime pfx from invitrogen.....

good luck...
I would prefer being perfectionist rather than a passionist in Research.

I always had an alternate hypothesis....

#5 Alisa

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Posted 14 May 2012 - 11:52 PM

i have done lot of SDMs and i never had failure untill now...your protocol looks ok, but your PCR cycle shdnt be 30. may be 15 shd be more than enough. after dpn digestion do a gel purification and transform. my usual primer design tool is Primer X.....i use Accuprime pfx from invitrogen.....

good luck...

Hello Veteran:
I've done 18cycles before, but I got no colonies.

Thanks for advises!

#6 GNANA

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Posted 15 May 2012 - 12:33 AM

Did you check on gel after PCR done with 18 cycles??. bcoz no colonies might also be due to lack of adequate competency in your competent cells. anyways doing 30 cycles on 12 kb vector is not recommended as your enzyme availability and fidelity for the entire 30 cycles is a matter of concern. even i you get positive SDMs there is also possibility of other mutations with your 30 cycle PCR. so i would suggest you to standardize your PCR to get a band in 15 cycles or around.
I would prefer being perfectionist rather than a passionist in Research.

I always had an alternate hypothesis....

#7 Alisa

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Posted 15 May 2012 - 09:30 PM

Did you check on gel after PCR done with 18 cycles??. bcoz no colonies might also be due to lack of adequate competency in your competent cells. anyways doing 30 cycles on 12 kb vector is not recommended as your enzyme availability and fidelity for the entire 30 cycles is a matter of concern. even i you get positive SDMs there is also possibility of other mutations with your 30 cycle PCR. so i would suggest you to standardize your PCR to get a band in 15 cycles or around.

Hi

I can't see band on my PCR gel, but

Stragene's guideline said that maybe can't visable. Is that true?

I also try to send plasmid( not mutation) to competent cells, and I got lots of colonies. Seems that my competent cells have no problem.



#8 GNANA

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Posted 16 May 2012 - 12:22 AM

Reg your competent cells taking up the plasmid doesnt mean it is enough competent to take up and process the nicked , and relaxed plasmid produced by the Linear amplification. so make your the efficiency of your competent cells if it is home made.

getting original plasmid suggests your Dpn digestion is not complete, so have a check over there as well.

Theoritically its true (strategenes guidelines) , but i personally dnt believe unless i see....anyways how you made sure your PCR worked??? did you see band with 30 cycle PCR?? how much PCR you run to check the band?? if it s just below 5% of total volume you may not see but i would suggest, after dpn digestion run the whole PCR and look for band, here you definitely shd see the band if your PCR worked, eventually you can do a gel purification and transformation of whole product or part of it depending on the band intensity....if not you still got to standardize your PCR rather than troubleshooting downstream.

good luck,
Gnana...
I would prefer being perfectionist rather than a passionist in Research.

I always had an alternate hypothesis....

#9 hln

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Posted 16 May 2012 - 11:36 AM

If you're getting that many mutant colonies, then either something is wrong with your miniprep (dirty or denatured DNA that resists DpnI digest) or your primers are binding in the wrong place. Try alternative primer design strategies, such as one described by Liu and Naismith, BMC Biotech 8:91.

#10 Alisa

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Posted 17 May 2012 - 01:01 AM

Reg your competent cells taking up the plasmid doesnt mean it is enough competent to take up and process the nicked , and relaxed plasmid produced by the Linear amplification. so make your the efficiency of your competent cells if it is home made.

getting original plasmid suggests your Dpn digestion is not complete, so have a check over there as well.

Theoritically its true (strategenes guidelines) , but i personally dnt believe unless i see....anyways how you made sure your PCR worked??? did you see band with 30 cycle PCR?? how much PCR you run to check the band?? if it s just below 5% of total volume you may not see but i would suggest, after dpn digestion run the whole PCR and look for band, here you definitely shd see the band if your PCR worked, eventually you can do a gel purification and transformation of whole product or part of it depending on the band intensity....if not you still got to standardize your PCR rather than troubleshooting downstream.

good luck,
Gnana...

I load 1/3 of my PCR product. Actually I am not sure my PCR works or not. I hope it's work! But I can't see band.
Thank you~

#11 Jason_LuYZ

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Posted 17 May 2012 - 03:43 AM

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

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 min

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

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.

#12 Alisa

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Posted 17 May 2012 - 07:58 PM


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

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 min

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

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.

HI

f-primer: ggatgcagaattccgacgtgactcaggatatgaag
r-primer: cttcatatcctgagtcacgtcggaattctgcatcc

this is my primer from stratgene primer design tool. But when I order primer from company, they gave me Tm just only 65℃.
I didn't do control!

Thanks for advise!

#13 Jason_LuYZ

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Posted 18 May 2012 - 11:28 PM



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

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 min

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

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.

HI

f-primer: ggatgcagaattccgacgtgactcaggatatgaag
r-primer: cttcatatcctgagtcacgtcggaattctgcatcc

this is my primer from stratgene primer design tool. But when I order primer from company, they gave me Tm just only 65℃.
I didn't do control!

Thanks for advis

Glad to see your primer and you should calculate the Tm by yourself, but not using the Tm provided by the primer-synthesis company.
Given you used Stratgene primer design tools, this may not have a severe problem, but I think the 12-17bp around both sides of your mutated is good principle.

What's more, you may give me a short sequence(often a complete sequence is more important) containning your mutaed genes or amino acids(should tell me the translate sequence and your DNA sequence), I may help you design 1-2 primers for your disgining. The primer designing programe I used is DNAstar software. A strategy for make mutagenesis may change the Codon seqence but not affecting your amino acids due to the degeneracy of codon. Sometimes I use this to reduce mispairing by influenced by the mutated nucleotide sequence.

Edited by Jason_LuYZ, 19 May 2012 - 12:08 AM.


#14 CAT

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Posted 19 June 2012 - 12:30 PM

Well, I do not know where goes wrong. But maybe, your DPnI enzyme did not work well and your template DNA (WT) are not digested completely?

I just followed the protocol. XL-blue, rather than DH5alpha, should be used after mutagenesis. You may try.

#15 anf_zahra

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Posted 29 July 2012 - 05:57 PM


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

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 min

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

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.


I am working on a site-directed mutagenesis, using pfu polymerase (Promega), and dpn1(NEB)


Here is my problem: My plasmid is 3.7kb big. Until now I can't figure out what's going wrong. I can not observe any bands in my gel(except faint band of primer dimers). also tried to transform after dpn1 digestion- no colonies to be seen.. ;(


i followed the protocol http://www.methodboo...pcr/pcrmut.html


but modified the cycle conditions as follows



1. 94 °C 2 min


2. 94 °C 30 sec
3. 65°C 30 sec(< 5°C less than my primers Tm-70 °C)


4. 72°C 2min/kb plasmid (so for my plasmid 7.5min)
5. 72°C 10min
Please suggest me if something is wrong with my method
Thanks in advance!!!

my topic : http://www.protocol-...cr-mutagenesis/







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