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Another primer dimers problem


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#16 Fluffy

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Posted 20 August 2011 - 08:45 PM

A control reaction is a DNA extract that MUST work with your primers (usually a cloned copy of your gene or a DNA extract that gives a good signal). When doing PCR you should include one positiv and one negative control in each run - means you PCR your samples + the postive control that is expected to work + a negative control that must be negative (usually just the mastermix and water instead of the DNA). This allows you to control your PCRs - i.e. you do not need to worry why your results are negative when the postive control fails - this means something was wrong with your PCR. If the negative control is positive you cannot trust your results as well because you have a DNA contamination somewhere.

Of course this is difficult when you have new primers and do not know if they work or not - have you only ordered one set of primers or have you more primer pairs to test?


Hello gebirgsziege,

Awesome thanks for the detailed explanation regarding controls. I am really fresh on this. I greatly appreciate your time in this.
It makes a lot of sense. I only have one set of primers but I will definitely order another set, my professor said the samethig too :D

Thanks dude!

#17 Fluffy

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Posted 20 August 2011 - 08:49 PM

As it's already been said by leelee, you are using way too much primers. No wonder you get dimers Posted Image Dilute your stock to 10uM and then use 0.5-1ul for your final 20ul PCR reaction. Also, leelee already explained why your buffer concentration is wrong, is the final concentration in the PCR reaction that is wrong, not the 10x buffer.

585bp should not be too hard to amplify. I'd definitely try the settings Adrian said, your current Annealing and Extension times are too long. In fact I'd do 30sec at 94C, 30sec at 55C, 30sec at 72C for 30-35 cycles.

I personally would repeat a gradient PCR lowering the primer concentration, changing the cycling times, and adjusting the buffer concentration to be right. Also check you MgCl2 concentration, and what final concentration invitrogen suggest. Which also reminds me, make sure the 10x polymerase buffer you are using doesn't have any MgCl2 already.

IF that doesn't work, then is time to think about redesigning primers. To which I would add: TA clone your product and cut it from there, rather than digest the PCR product directly. But then that's personal preferences.

Oh! And I think your dNTPs concentration is fine but you might have to alter it if you change the MgCl2 concentration, but I wouldnt worry to much about that just now.

Good luck, and let us know if/when you get your product! Posted Image



Hi Almost a doctor,

Great!!

It really makes alot of sense to me now. I will try all the ways all of you mentioned and let you know if anything happens with me (cross your fingers hehe). Honestly you are very helpful and I will try everything mentioned since I did not have much knowledge about all the troubleshooting in regards to PCRs.

I will let my professor know about your idea regarding PCR product digestion and TA cloning for sure Posted Image

Thanks again for everything!!!!

Edited by yasamino, 20 August 2011 - 08:50 PM.


#18 Adrian K

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Posted 21 August 2011 - 01:43 AM

Hello Adrian,

Great!!
I am sorry I mentioned that I used Invitrogen. Well I did before, but that specific run I used a bioshop kit which I buy from our science store. It is 25mM. I am sorry Posted Image

I will take your steps of PCR cycles into consideration. This is awesome!
Thanks again!!


Dear yasamino,
Hahahaha... If you want people to help you accurately, please provide accurate information next time. No worries,no body will copy your PCR mastermix formula because almost all the people who responded to you here have their own mastermixes which worked for them... Posted Image

Can you show your recent gel picture?
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434

#19 Fluffy

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Posted 24 August 2011 - 09:10 AM

Hello All,

I have attached a gel image of the recent PCR I did using the following conditions:

10X buffer-2ul
25mM MgCl2-1.6ul
10mM dNTP-0.75ul
10uM Forward Primer-0.5ul
10uM Reverse Primer-0.5ul
Taq-0.3ul
cDNA template-4ul (for concentration check first post of mine)
dH2O-add up to 20ul

PCR conditios
94C-4min
94C-45sec
Anneal (I did a gradient of 52-62C)-30sec
72C-30sec go to 2 repeat 30 times
72C-5min
4-for ever

My product size is as mentioned earlier in posts 585 bp. The gel image has 6 products that are very faint and they correspond to the following temps

From left
1-53.7
2-54.8
3-56.3
4-58.0
5-59.4
6-60.5
Note: the first lane is a 1kb ladder, and between each one i skipped a lane.

So there are really faint bands, and 2 seems to be the brightest but looks like a smear. This sort of gives me hope before I did not see anything just primer dimers, but I obviously have to work more on it. Should I increase cDNA template? Is my cDNA contaminated or degraded or..? What can I do for even better results? I was thinking of trying 55 as annealing temp.?

Thanks ALL and I GREATLY APPRECIATE all the help!!!!!
Yasamino08.24.2011 UB2 PCR w changes 08.23.2011.jpg

#20 Adrian K

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Posted 26 August 2011 - 11:02 AM

Dear Yasamino,
There are six lanes, For band number 2 &3, there are 2 bands.
Maybe I am wrong, but I do not find that your band sizes were corresponded with the size you wanted. it is like 1.2kb and 1.8kb. Which brand of 1kb ladder you use, can provide the catalog number as well as a reference?

Let's say the band you wanted is the bottom band, I suggest you directly just pool all your samples, run it in agarose gel, do a gel purification and elution and finally do a cloning into your desired vector. There is no need to waste so much time on PCR optimization.

Is hard to say in this stage that whether your cDNA is degraded or whether you should increase it. I suggest you try to use your genomic DNA as control and see if there is any differences, provided your primer was not designed on the splicing site. Also, addition of Betaine or DMSO might help to eliminate the unspecificity binding and perhaps reduce smear. This is not the issue of primer dimmer.
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434

#21 Fluffy

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Posted 26 August 2011 - 11:46 AM

Dear Yasamino,
There are six lanes, For band number 2 &3, there are 2 bands.
Maybe I am wrong, but I do not find that your band sizes were corresponded with the size you wanted. it is like 1.2kb and 1.8kb. Which brand of 1kb ladder you use, can provide the catalog number as well as a reference?

Let's say the band you wanted is the bottom band, I suggest you directly just pool all your samples, run it in agarose gel, do a gel purification and elution and finally do a cloning into your desired vector. There is no need to waste so much time on PCR optimization.

Is hard to say in this stage that whether your cDNA is degraded or whether you should increase it. I suggest you try to use your genomic DNA as control and see if there is any differences, provided your primer was not designed on the splicing site. Also, addition of Betaine or DMSO might help to eliminate the unspecificity binding and perhaps reduce smear. This is not the issue of primer dimmer.


Hello Adrian,
Thanks for your reply. Much appreciated. I am sorry for my questions, I noticed you mentioned you work long hours in the lab. I am really sorry if I am being a bother.

The ladder I am using is a 1kb ladder from Invitrogen and its Cat # is 15615-016.
Also I do notice two bands in each lane, you are right, but I thought the second band on the bottom would be my right size, since the in the ladder the bright band is 500bp and right above it would be close to 585bp?
OK so you think gel purification is a better idea than PCR clean up using the PCR sample itself, rather than gel?
And when you say genomic DNA do you mean I order a DNA extract of my gene only and use that as a positive control with my primers?

I was thinking of using the DMSO and or formamide today but I thought I should receive some feedback from the forum :D

But really thanks alot, you are great help :D

#22 Adrian K

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Posted 27 August 2011 - 12:53 AM


Dear Yasamino,
There are six lanes, For band number 2 &3, there are 2 bands.
Maybe I am wrong, but I do not find that your band sizes were corresponded with the size you wanted. it is like 1.2kb and 1.8kb. Which brand of 1kb ladder you use, can provide the catalog number as well as a reference?

Let's say the band you wanted is the bottom band, I suggest you directly just pool all your samples, run it in agarose gel, do a gel purification and elution and finally do a cloning into your desired vector. There is no need to waste so much time on PCR optimization.

Is hard to say in this stage that whether your cDNA is degraded or whether you should increase it. I suggest you try to use your genomic DNA as control and see if there is any differences, provided your primer was not designed on the splicing site. Also, addition of Betaine or DMSO might help to eliminate the unspecificity binding and perhaps reduce smear. This is not the issue of primer dimmer.


Hello Adrian,
Thanks for your reply. Much appreciated. I am sorry for my questions, I noticed you mentioned you work long hours in the lab. I am really sorry if I am being a bother.

The ladder I am using is a 1kb ladder from Invitrogen and its Cat # is 15615-016.
Also I do notice two bands in each lane, you are right, but I thought the second band on the bottom would be my right size, since the in the ladder the bright band is 500bp and right above it would be close to 585bp?
OK so you think gel purification is a better idea than PCR clean up using the PCR sample itself, rather than gel?
And when you say genomic DNA do you mean I order a DNA extract of my gene only and use that as a positive control with my primers?

I was thinking of using the DMSO and or formamide today but I thought I should receive some feedback from the forum Posted Image

But really thanks alot, you are great help Posted Image


Haha, don't don't apologize, is not your fault. When I plan my work, I forget that almost the next whole week is my country's holiday, and I can't use most of the instruments. Most of the staff will be absent, and thus the office closed.. I got no choice but to stay back for "lab marathon".

Yes, gel-excised purification is always better as it only cut out and isolate the band you wanted. Sorry for make you confuse in my previous post. The shortcut (a.k.a cheat) I was trying to say is you can use a PCR clean up spin column, load all of the remaining PCR products from your gradient PCR (would be roughly 15ul from each tube times 6 equals to around 90ul total volume), do a clean up and elute the product (using around 40ul elution buffer). After the elution, load all the eluted product onto agarose gel, run it, and gel-excised-purified the band you required (your case, cut only the 585bp band).

Alternatively, you just load all your ~90ul product onto the agarose gel, run the electrophoresis and proceed with gel-excised-purification for the 585bp band. During your elution step, try elute with less than 40ul elution buffer (30ul would be even better as it 3x concentrated your initial volume of ~90ul). Use the product for your cloning purposes.

My suggestions is mean for you to speed up your work rather than just focus on PCR optimization. Those were the few tricks I use for my work, yet to fail me for the almost 30 different clones I did. Just imagine the time you required for optimizing 30 independent PCR, unless you are using for endpoint detection.

However for your cloning purposes, as phage434 mentioned earlier, you might not get it done due to those RE required extra 5-6 bases. I suggest you do a TA / Blunt end cloning first, send for sequencing and after your new primers arrived, you PCR it again using the cloned plasmid, so that you do not need to use your cDNA again.

For the PCR addictives, I never heard of using "formamide". The addictives commonly used is betaine, DMSO and BSA.

Hope this helps.

Regards,
Adrian
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434

#23 Fluffy

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Posted 13 September 2011 - 05:30 PM



Dear Yasamino,
There are six lanes, For band number 2 &3, there are 2 bands.
Maybe I am wrong, but I do not find that your band sizes were corresponded with the size you wanted. it is like 1.2kb and 1.8kb. Which brand of 1kb ladder you use, can provide the catalog number as well as a reference?

Let's say the band you wanted is the bottom band, I suggest you directly just pool all your samples, run it in agarose gel, do a gel purification and elution and finally do a cloning into your desired vector. There is no need to waste so much time on PCR optimization.

Is hard to say in this stage that whether your cDNA is degraded or whether you should increase it. I suggest you try to use your genomic DNA as control and see if there is any differences, provided your primer was not designed on the splicing site. Also, addition of Betaine or DMSO might help to eliminate the unspecificity binding and perhaps reduce smear. This is not the issue of primer dimmer.


Hello Adrian,
Thanks for your reply. Much appreciated. I am sorry for my questions, I noticed you mentioned you work long hours in the lab. I am really sorry if I am being a bother.

The ladder I am using is a 1kb ladder from Invitrogen and its Cat # is 15615-016.
Also I do notice two bands in each lane, you are right, but I thought the second band on the bottom would be my right size, since the in the ladder the bright band is 500bp and right above it would be close to 585bp?
OK so you think gel purification is a better idea than PCR clean up using the PCR sample itself, rather than gel?
And when you say genomic DNA do you mean I order a DNA extract of my gene only and use that as a positive control with my primers?

I was thinking of using the DMSO and or formamide today but I thought I should receive some feedback from the forum Posted Image

But really thanks alot, you are great help Posted Image


Haha, don't don't apologize, is not your fault. When I plan my work, I forget that almost the next whole week is my country's holiday, and I can't use most of the instruments. Most of the staff will be absent, and thus the office closed.. I got no choice but to stay back for "lab marathon".

Yes, gel-excised purification is always better as it only cut out and isolate the band you wanted. Sorry for make you confuse in my previous post. The shortcut (a.k.a cheat) I was trying to say is you can use a PCR clean up spin column, load all of the remaining PCR products from your gradient PCR (would be roughly 15ul from each tube times 6 equals to around 90ul total volume), do a clean up and elute the product (using around 40ul elution buffer). After the elution, load all the eluted product onto agarose gel, run it, and gel-excised-purified the band you required (your case, cut only the 585bp band).

Alternatively, you just load all your ~90ul product onto the agarose gel, run the electrophoresis and proceed with gel-excised-purification for the 585bp band. During your elution step, try elute with less than 40ul elution buffer (30ul would be even better as it 3x concentrated your initial volume of ~90ul). Use the product for your cloning purposes.

My suggestions is mean for you to speed up your work rather than just focus on PCR optimization. Those were the few tricks I use for my work, yet to fail me for the almost 30 different clones I did. Just imagine the time you required for optimizing 30 independent PCR, unless you are using for endpoint detection.

However for your cloning purposes, as phage434 mentioned earlier, you might not get it done due to those RE required extra 5-6 bases. I suggest you do a TA / Blunt end cloning first, send for sequencing and after your new primers arrived, you PCR it again using the cloned plasmid, so that you do not need to use your cDNA again.

For the PCR addictives, I never heard of using "formamide". The addictives commonly used is betaine, DMSO and BSA.

Hope this helps.

Regards,
Adrian


Hey Adrian,

I have been sick for some time, and did not have a productive 2 weeks.
But Thanks for your latest reply. I will do TOPO cloning so that is great. And I will gene cut my PCR, I believe that is best option for me.
Thanks,
Yasamino :D

#24 Adrian K

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Posted 13 September 2011 - 08:01 PM

Hi there Yasamino,
All the best to you. Good luck and take good care of your health.

Regards,
Adrian
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434




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