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Problems with Quick change lightning multi site directed mutagenesis kit


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#1 Adnan

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Posted 18 September 2009 - 07:13 AM

I am using quick change lightning multi site directed mutagenesis kit from stratagene, i want to mutate 5nt , my plasmid+insert size is about 6kb, i strictly follow the protocol but it always fails..........its annoying coz i have almost spend 1.5 month on fighting with this kit but it never worked............i changed the PCR parameters and increased time from 30sec per kb to 1min per kb, also increased the annealing temperature to 65 instead of normal 55c........every time i do it on the pursuit that this time i will get colonies but its always the same.............What can be wrong? i use every thing as specified in the kit and i dnt move an ioata from it..........
my forward primer is GAG ACG AAG GAG AAC CTA TAT TTC CAG GGC GAT TTA TTA TTG

and my reaverse primer is CAA TAA TAA ATC GCC CTG GAA ATA TAG GTT CTC CTT CGT CTC .....................

the mutations are balanced as they are exactly in the middle.......primer has no problem of self annealing, complimentarity and hairpin formation...........

how can i know that my pcr worked or not? after using Dpn1 enzyme can i view DNA on the gel?

i was suspecious about competency of the cells i used wild dna as a control and it gave me just 8 colonies for the whole reaction.....so i quit to use XL10 Gold and now instead i used BL21Gold......in ist reaction there were no colonies so i checked its competency with wild dna and it was satisfactory, it gave 33 colonies instead of 50..........have done a colony PCR just to confirm transformation but on the gel nothing is visible...........so again a lot of questions are raised.......can anybody plz tell me how to make this kit work? am fed up of constant failures .......

#2 Warren

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Posted 18 September 2009 - 08:43 AM

I am using quick change lightning multi site directed mutagenesis kit from stratagene, i want to mutate 5nt , my plasmid+insert size is about 6kb, i strictly follow the protocol but it always fails..........its annoying coz i have almost spend 1.5 month on fighting with this kit but it never worked............i changed the PCR parameters and increased time from 30sec per kb to 1min per kb, also increased the annealing temperature to 65 instead of normal 55c........every time i do it on the pursuit that this time i will get colonies but its always the same.............What can be wrong? i use every thing as specified in the kit and i dnt move an ioata from it..........
my forward primer is GAG ACG AAG GAG AAC CTA TAT TTC CAG GGC GAT TTA TTA TTG

and my reaverse primer is CAA TAA TAA ATC GCC CTG GAA ATA TAG GTT CTC CTT CGT CTC .....................

the mutations are balanced as they are exactly in the middle.......primer has no problem of self annealing, complimentarity and hairpin formation...........

how can i know that my pcr worked or not? after using Dpn1 enzyme can i view DNA on the gel?

i was suspecious about competency of the cells i used wild dna as a control and it gave me just 8 colonies for the whole reaction.....so i quit to use XL10 Gold and now instead i used BL21Gold......in ist reaction there were no colonies so i checked its competency with wild dna and it was satisfactory, it gave 33 colonies instead of 50..........have done a colony PCR just to confirm transformation but on the gel nothing is visible...........so again a lot of questions are raised.......can anybody plz tell me how to make this kit work? am fed up of constant failures .......


I am guessing by some of the things you have said, that molecular biology is something new to you -- do you have any colleagues in the lab to run through it with? You are doing some major things wrong, if I read correctly, just in what you have said so far, that makes me think there could be some other things wrong. Firstly you always have to check PCR, are you assuming it works without ever running it on a gel? Secondly, BL21 cells of any variety are not for cloning, but for expression, you should not be using these! Maybe run down some ore details and we can give you more hints....Warren..

#3 Adnan

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Posted 18 September 2009 - 03:49 PM

Dear Warren,
Yeh am new to Molecular biology, I have colleagues in the Lab at ist they were saying you are doing something wrong, one of them who was the most experienced ran a parrallel experiment with me just to check wat is wrong, me or something else? the result was the same.....I was doing it according to the protocol given with the kit........it was not working then they told me to change the parameters of PCR so i increased the annealing temperature and the extension temperature to get more specific product but it was of no use ......as regards running of DNA on gel before transformation, i was doing it but they stopped me because of their faith in the kit to be the most accurate tool as they have used it several times and was always succesfull......thats y am not doing the control reaction.
when it failed no one was believing in what happened.......I understand its shameful coz dealing with a kit is not rocket science as u have to follow strictly the instructions given ........Ok let us assume that PCR did not work then y the transformation was not working with wild type plasmid, that was extracted with a kit (Qiagen mini prep), double checked on gel......undigested plasmid and digested with restriction enzyme so as to know that the gene of intrest is present......and was confirmed to be present...if the wild type plasmid (without mutation) is not transforming is not a problem of competency?
I am using BL21-Gold (DE3)pLysS competent cells and these are the orignal cells from which i extracted the plasmid and now am trying to mutate and transform.........These cells come with mutagenesis kit of stratagene..........at ist attempt it gave me blank plates but on second attempt it gave me 33 colonies......so before extracting Plasmid DNA from it i tried a colony PCR as a short cut to confirm but it failed........moreover the problem is that when you transform these cells with Dpn1 treated single stranded DNA it gives no colonies and when transformed with double stranded DNA (which is not treated with Dpn1) it give colonies..............
I am using plasmid pKK233-2 which has my gene of interest and all i have to do is to extract, mutate and retransform (Express it)........

SO FAR

In so many attempts so far i got one colony with XL10Gold competent cells......which might not have mutated as sequencing will confirm its reality although on re plating and culture in selective media it grew but the wild type also has amp resistant gene so??????????.....The other weired thing that happened was that the third time when i was doing transformation nothing was found on plates but there was growth in LB+ampicillin, and replating and culturing there were bundles of colonies but it might have the same status as the ist one..........I am quite sure that some of the parental dsDNA might have left in the process and it got transformed as it is confirmed by BL21GOld cells.......so the net is zero........Thats what am worried about......and its reality.
Now my all concern is about PCR......I ran ssDNA on gel (5ul) but nothing was visible and i think it was unvisible coz it was single stranded......is it possible to see single stranded DNA on Gel?
Ok next time i will run double stranded DNA on gel before adding Dpn1 to it but there is possibility of false result because its not necessary that the DNA am visualizing on the gel is the mutated one as there might be some parental DNA left behind..........I think the only way to check your result is after transformation and in the mean time its my problem...........
if single stranded DNA is visible on gel then its ok coz then there will be no traces of parental DNA as it will be assumed to completely digested by Dpn1....or can i confirm my PCR product by some other means?
Thanking you in anticipation and waiting for your kind suggestion...
Regards,


I am guessing by some of the things you have said, that molecular biology is something new to you -- do you have any colleagues in the lab to run through it with? You are doing some major things wrong, if I read correctly, just in what you have said so far, that makes me think there could be some other things wrong. Firstly you always have to check PCR, are you assuming it works without ever running it on a gel? Secondly, BL21 cells of any variety are not for cloning, but for expression, you should not be using these! Maybe run down some ore details and we can give you more hints....Warren..
[/quote]

#4 Warren

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Posted 18 September 2009 - 07:11 PM

Dear Warren,
Yeh am new to Molecular biology, I have colleagues in the Lab at ist they were saying you are doing something wrong, one of them who was the most experienced ran a parrallel experiment with me just to check wat is wrong, me or something else? the result was the same.....I was doing it according to the protocol given with the kit........it was not working then they told me to change the parameters of PCR so i increased the annealing temperature and the extension temperature to get more specific product but it was of no use ......as regards running of DNA on gel before transformation, i was doing it but they stopped me because of their faith in the kit to be the most accurate tool as they have used it several times and was always succesfull......thats y am not doing the control reaction.
when it failed no one was believing in what happened.......I understand its shameful coz dealing with a kit is not rocket science as u have to follow strictly the instructions given ........Ok let us assume that PCR did not work then y the transformation was not working with wild type plasmid, that was extracted with a kit (Qiagen mini prep), double checked on gel......undigested plasmid and digested with restriction enzyme so as to know that the gene of intrest is present......and was confirmed to be present...if the wild type plasmid (without mutation) is not transforming is not a problem of competency?
I am using BL21-Gold (DE3)pLysS competent cells and these are the orignal cells from which i extracted the plasmid and now am trying to mutate and transform.........These cells come with mutagenesis kit of stratagene..........at ist attempt it gave me blank plates but on second attempt it gave me 33 colonies......so before extracting Plasmid DNA from it i tried a colony PCR as a short cut to confirm but it failed........moreover the problem is that when you transform these cells with Dpn1 treated single stranded DNA it gives no colonies and when transformed with double stranded DNA (which is not treated with Dpn1) it give colonies..............
I am using plasmid pKK233-2 which has my gene of interest and all i have to do is to extract, mutate and retransform (Express it)........

SO FAR

In so many attempts so far i got one colony with XL10Gold competent cells......which might not have mutated as sequencing will confirm its reality although on re plating and culture in selective media it grew but the wild type also has amp resistant gene so??????????.....The other weired thing that happened was that the third time when i was doing transformation nothing was found on plates but there was growth in LB+ampicillin, and replating and culturing there were bundles of colonies but it might have the same status as the ist one..........I am quite sure that some of the parental dsDNA might have left in the process and it got transformed as it is confirmed by BL21GOld cells.......so the net is zero........Thats what am worried about......and its reality.
Now my all concern is about PCR......I ran ssDNA on gel (5ul) but nothing was visible and i think it was unvisible coz it was single stranded......is it possible to see single stranded DNA on Gel?
Ok next time i will run double stranded DNA on gel before adding Dpn1 to it but there is possibility of false result because its not necessary that the DNA am visualizing on the gel is the mutated one as there might be some parental DNA left behind..........I think the only way to check your result is after transformation and in the mean time its my problem...........
if single stranded DNA is visible on gel then its ok coz then there will be no traces of parental DNA as it will be assumed to completely digested by Dpn1....or can i confirm my PCR product by some other means?
Thanking you in anticipation and waiting for your kind suggestion...
Regards,


I am guessing by some of the things you have said, that molecular biology is something new to you -- do you have any colleagues in the lab to run through it with? You are doing some major things wrong, if I read correctly, just in what you have said so far, that makes me think there could be some other things wrong. Firstly you always have to check PCR, are you assuming it works without ever running it on a gel? Secondly, BL21 cells of any variety are not for cloning, but for expression, you should not be using these! Maybe run down some ore details and we can give you more hints....Warren..


OK, I had to go look at this kit to make sense of some of what you are trying to do, and right off the bat I see a couple giant problems. You are describing protocols from two different kits -- the multi-site kit (which is where you will end up with single stranded DNA) and the regular quick change kit (which is where the primers you describe would be actually used). Its important to figure out which you are using, as they are different. Based on the primers you designed (complementary and both containing the mutation) you do NOT use these with the multi-site kit. If you are indeed using the multi-site kit, you only want to use ONE of these primers, NOT BOTH. We need to figure this out first, as you are describing two different kits. A couple more things:

DO NOT use BL21 cells for cloning!!! You will use these AFTER you have identified your correct clone from normal competent cells, then retransform into BL21s. Trust me on this, use your other cells.

I can almost guarantee your cells are competent. The PROPER way to test competency (if you REALLY think this is a problem, highly unlikely!) is to use the control plasmid, pUC18 that comes with the kit. I don't like to waste cells like this but you can do this to convince yourself if you must. The colonies you were seeing before were from the original plasmid.

You have described a protocol that is part multi-site/part regular quick change and that is your problem....which kit are you actually using????

Warren..

#5 Warren

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Posted 18 September 2009 - 07:41 PM

one other thing, you CAN see single stranded DNA on a gel....it migrates differently than double-stranded, but you can definitely see it.....however, you are not going to get single stranded DNA by using BOTH of those primers with the multi-site kit! :D Warren..

#6 Adnan

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Posted 20 September 2009 - 03:24 PM

Dear Warren,
I am using quick change multi site directed mutagenesis kit.........and am following its protocol only, nt a mixed protocol.
I just used BL21gold pLYsS competent cells as an extra transformation reaction, u may consider it as a control......

Have two issues with what u suggested....

1. How can u say that two primers (forward and reverse) are not used in multisite directed mutagenesis kit? have u gone through its protocol? coz it asks for both primers nt one and also if u use one primer........let us assume its so then what happens? the plasmid is denatured.......the next step is annealing,with one primer (it will anneal to one strand of parental DNA) whats the destiny of the second complementary strand of parental DNA?will it pile up unmutated............???????? decreasing the efficiency of mutagenesis

2. BL21 gold cells that am using as a secondry option comes with mutagenesis kit, they are also competent cells, ppl in our lab used and are using it........yes they are very good for expression .........
Have searched for running DNA on the gel in this specific case and they say it may or may not be visible on the gel if it is used in 10ul quantity (from mutagenesis reaction) any how proceed to the next step and add Dpn1 to digest any parental dsDNA.......and then transformation.(so u have to rely on transformation)........
I think i wrote too much and was nt clear and understandable? was it?

#7 Adnan

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Posted 20 September 2009 - 03:27 PM

One thing more that i will add to is that DNA in this case is not fully single stranded but in nicked form.....and is transformed at this stage into the bacterium

#8 Warren

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Posted 20 September 2009 - 08:32 PM

Dear Warren,
I am using quick change multi site directed mutagenesis kit.........and am following its protocol only, nt a mixed protocol.
I just used BL21gold pLYsS competent cells as an extra transformation reaction, u may consider it as a control......

Have two issues with what u suggested....

1. How can u say that two primers (forward and reverse) are not used in multisite directed mutagenesis kit? have u gone through its protocol? coz it asks for both primers nt one and also if u use one primer........let us assume its so then what happens? the plasmid is denatured.......the next step is annealing,with one primer (it will anneal to one strand of parental DNA) whats the destiny of the second complementary strand of parental DNA?will it pile up unmutated............???????? decreasing the efficiency of mutagenesis

2. BL21 gold cells that am using as a secondry option comes with mutagenesis kit, they are also competent cells, ppl in our lab used and are using it........yes they are very good for expression .........
Have searched for running DNA on the gel in this specific case and they say it may or may not be visible on the gel if it is used in 10ul quantity (from mutagenesis reaction) any how proceed to the next step and add Dpn1 to digest any parental dsDNA.......and then transformation.(so u have to rely on transformation)........
I think i wrote too much and was nt clear and understandable? was it?


Yes, as a matter of fact, I have gone through the protocol. It asks for more than one primer because its designed for MULTIPLE mutations in a single rxn, hence the name MULTI-SITE. Read the beginning of the manual. It quite CLEARLY states that multiple primers are designed to be attached the the SAME STRAND, to make multiple mutations at different sites. And your synopsis of what happens during the thermocycling (not PCR, but linear amplification) is exactly what is says will happen in the manual. The parental DNA will not "pile up" but will remain as whatever you added to the reaction, that is digested by the enzyme you add at the end.

This is why you end up with SINGLE-STRANDED DNA at the end. Nicked double strands are NOT single stranded DNA. Once again, the reason the protocol mentions multiple primers is because it is designed to mutate multiple areas at the same time. If you are only going to make a mutation at one site, you should only use ONE primer. You can either believe me, read the beginning of the manual where it talks very clearly about this (not just the protocol, the whole manual, you can get it online), or you can keep making the same mistake. That is where you problem is. You designed primers to be used for the regular kit, and are trying to follow the protocol and use the reagents for the multi-site kit. You have two choices -- use the regular kit and both of your primers -- use the multi-site kit and one of your primers.

I don't know if you will have enough ssDNA to see on a gel. All I am saying there is that single-stranded DNA is definitely visible on a gel, provided there is enough of it. If you have enough parental template to see on a gel, and you do things correctly, you should have enough ssDNA to see as well.

According to Stratagene, BL21 cells do not come with these kits, but I suppose you may have gotten some special kit that included components for expression. However, BL21 cells are not supposed to be used for cloning. They are not another option. I am sure there are other cells in your lab for cloning, BL21s are not them. Read the manuals on BL21s they will tell you do NOT do the cloning in them. You make your clones, and when you have identified the correct clone, then you transform into BL21 for expression. BL21s do not have the genetic make-up to be good for cloning.

If you do not believe the things I have written, print them out and show them to some post-docs in your lab, preferably ones who have used the MULTI-SITE kit and have used BL21s before. I am not sure if there is a language barrier here, but your response is coming across as if you are trying to argue with me, which I am sure senior members of your lab won't appreciate. I took the time to read the manual, figured out your problem for you, and now it sounds like you are trying to tell me I am wrong. I am not trying to brag, but I have years of experience with molecular biology, I am not just spouting off, I was trying to help you. I identified the problem, you can fix it, or you can argue with me about it, but I am sure anyone on this group who wants to read the manual from Statagene will tell you I am right. Good luck, Warren..

#9 Adnan

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Posted 21 September 2009 - 01:29 PM

Dear Warren,
I appologise for it, English is nt my native language and thats y have difficulty in expressing what i actually mean........i dnt want to argue with you and i cant........I just meant to ask and make sure as vat am going to do in the next step............i always consider it good to be sure coz am new to the field and i need guidence and at times some explanation from experienced people like you...infact i was waiting for you to make me sure,now i am so i will proceed now and will work this time.....coz the fault is figured out,thanks a lot for that ..........i hope u will guide me in future......

Best Regards,
Adnan







Yes, as a matter of fact, I have gone through the protocol. It asks for more than one primer because its designed for MULTIPLE mutations in a single rxn, hence the name MULTI-SITE. Read the beginning of the manual. It quite CLEARLY states that multiple primers are designed to be attached the the SAME STRAND, to make multiple mutations at different sites. And your synopsis of what happens during the thermocycling (not PCR, but linear amplification) is exactly what is says will happen in the manual. The parental DNA will not "pile up" but will remain as whatever you added to the reaction, that is digested by the enzyme you add at the end.

This is why you end up with SINGLE-STRANDED DNA at the end. Nicked double strands are NOT single stranded DNA. Once again, the reason the protocol mentions multiple primers is because it is designed to mutate multiple areas at the same time. If you are only going to make a mutation at one site, you should only use ONE primer. You can either believe me, read the beginning of the manual where it talks very clearly about this (not just the protocol, the whole manual, you can get it online), or you can keep making the same mistake. That is where you problem is. You designed primers to be used for the regular kit, and are trying to follow the protocol and use the reagents for the multi-site kit. You have two choices -- use the regular kit and both of your primers -- use the multi-site kit and one of your primers.

I don't know if you will have enough ssDNA to see on a gel. All I am saying there is that single-stranded DNA is definitely visible on a gel, provided there is enough of it. If you have enough parental template to see on a gel, and you do things correctly, you should have enough ssDNA to see as well.

According to Stratagene, BL21 cells do not come with these kits, but I suppose you may have gotten some special kit that included components for expression. However, BL21 cells are not supposed to be used for cloning. They are not another option. I am sure there are other cells in your lab for cloning, BL21s are not them. Read the manuals on BL21s they will tell you do NOT do the cloning in them. You make your clones, and when you have identified the correct clone, then you transform into BL21 for expression. BL21s do not have the genetic make-up to be good for cloning.

If you do not believe the things I have written, print them out and show them to some post-docs in your lab, preferably ones who have used the MULTI-SITE kit and have used BL21s before. I am not sure if there is a language barrier here, but your response is coming across as if you are trying to argue with me, which I am sure senior members of your lab won't appreciate. I took the time to read the manual, figured out your problem for you, and now it sounds like you are trying to tell me I am wrong. I am not trying to brag, but I have years of experience with molecular biology, I am not just spouting off, I was trying to help you. I identified the problem, you can fix it, or you can argue with me about it, but I am sure anyone on this group who wants to read the manual from Statagene will tell you I am right. Good luck, Warren..
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#10 Warren

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Posted 21 September 2009 - 07:22 PM

Well, let us know if it works! Good luck! Warren..

Dear Warren,
I appologise for it, English is nt my native language and thats y have difficulty in expressing what i actually mean........i dnt want to argue with you and i cant........I just meant to ask and make sure as vat am going to do in the next step............i always consider it good to be sure coz am new to the field and i need guidence and at times some explanation from experienced people like you...infact i was waiting for you to make me sure,now i am so i will proceed now and will work this time.....coz the fault is figured out,thanks a lot for that ..........i hope u will guide me in future......

Best Regards,
Adnan



#11 Adnan

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Posted 24 September 2009 - 01:32 AM

Dear warren,

This time i got 6 colonies in total for one reaction and 13 colonies for the second reaction........The colony number is still very low, in the kit they say you must expect 10-1000 colonies depending on the number and nature of primers and base composition of DNA.........if this colony number corresponds to the whole reaction then its not bad i think.....what do you say?

on each plate i plated 250ul of transformation reaction.......(each transformation reaction was plated on 3 plates)




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