weird PCR results!?

[coralfangs]

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yesterday, i got these plasmids and primers from a company,
i ran them, only #3 and #5 plasmid/primer sets worked
today, i re-ran everything, only #2 and #4 plasmid/primer sets worked
(no, i didn't label them incorrectly becuz the sizes do correspond to the expected sizes)

what could happen here?
the only explanation i can come up with is that i didn't get enough plasmid at all (10pg per uL) and only about 10 uL in total for each plasmid, for #3 and #5 today, i had to spin several times just to get enough for my reaction

what else could have happened?

 

[ChristianJames1]

you really dont need much template DNA at all to get a product. did you use the same annealing temps both times? my PI told me the other day that PCR is more of an art than a science. sometimes you just have to fiddle with things (salt concentrations, DNA concentrations etc) and eventually you get them to work...most of the time you dont even know what you changed. good luck.

i might try lowering the annealing temp a couple degrees...maybe to 50 or 55?

 

[neuroprotector]

yesterday, i got these plasmids and primers from a company,
i ran them, only #3 and #5 plasmid/primer sets worked
today, i re-ran everything, only #2 and #4 plasmid/primer sets worked
(no, i didn't label them incorrectly becuz the sizes do correspond to the expected sizes)

what could happen here?
the only explanation i can come up with is that i didn't get enough plasmid at all (10pg per uL) and only about 10 uL in total for each plasmid, for #3 and #5 today, i had to spin several times just to get enough for my reaction

what else could have happened?

I guess we'll be seeing you in Stockholm.

 

[nonesuchgirl]

Are you the one that left their plasmids out overnight?

 

[coralfangs]

ive been using the same pcr protocol though
same temp., same duration, same # of cycles, etc

 

[coralfangs]

Are you the one that left their plasmids out overnight?

yes
the very same plasmids
hahaha

but.... that shouldn't explain why suddenly #2 and #4 work after leaving them in the ice bucket overnight

 

[nonesuchgirl]

yes
the very same plasmids
hahaha

😛 At least you're consistent.

 

[Tropicana100]

yesterday, i got these plasmids and primers from a company,
i ran them, only #3 and #5 plasmid/primer sets worked
today, i re-ran everything, only #2 and #4 plasmid/primer sets worked
(no, i didn't label them incorrectly becuz the sizes do correspond to the expected sizes)

what could happen here?
the only explanation i can come up with is that i didn't get enough plasmid at all (10pg per uL) and only about 10 uL in total for each plasmid, for #3 and #5 today, i had to spin several times just to get enough for my reaction

what else could have happened?

If you only have 10uL of plasmid from a company, you definitely need to back them up before doing anything. After you transform, miniprep and spec your DNA you will have plenty to use for PCR. I would use 10-100 ng/ul of plasmid in your PCR rxn since you don't need much plasmid at all. Also, I would do 3-4 PCR rxns for each plasmid then you can pool them together before you run your gel, that way you get nice and strong bands. Make sure you do a plasmid-only control when you run your gel in case it overlaps with your amplified fragments. One more idea is to double check the Tm's of your primers. Use an annealing temp about 5 deg C below the Tm's.

 

[jr doctor in sd]

hmmm this is really weird...and all the proteins are the same despite size?

how are 2 and 4 related in size to 3 and 5?

Only thing that I could think of is maybe 2/4 didnt have enough DNA day 1...and day two you unconsciously put alot more in 2/4 than 3/5 and so 2/4 showed out well and 3/5 wasnt a strong enough signal haha.

Ive only done PCR so far a few times..and no problems yet but yes meticulously using my timer and having my eye on the temperature

 

[coralfangs]

i wished i couldve backed up
my lab lacks most of the basic mol bio equipment
its a clinical lab

and for pcr, i do it in an automated pcr machine, so there couldn't be any problem

 

[themule]

hmmm this is really weird...and all the proteins are the same despite size?

how are 2 and 4 related in size to 3 and 5?

Only thing that I could think of is maybe 2/4 didnt have enough DNA day 1...and day two you unconsciously put alot more in 2/4 than 3/5 and so 2/4 showed out well and 3/5 wasnt a strong enough signal haha.

Ive only done PCR so far a few times..and no problems yet but yes meticulously using my timer and having my eye on the temperature

DNA fragments from PCR not proteins.

You really don't need much to "back up" the plasmids. Some LB media, plates, E. coli strain (DH5alpha), antibiotic. Really that's about it. There is some miscellaneous equipment but you should be able to make some of it if not all. PM me if you want further instructions.

TM

 

[Foghorn]

If you only have 10uL of plasmid from a company, you definitely need to back them up before doing anything. After you transform, miniprep and spec your DNA you will have plenty to use for PCR. I would use 10-100 ng/ul of plasmid in your PCR rxn since you don't need much plasmid at all. Also, I would do 3-4 PCR rxns for each plasmid then you can pool them together before you run your gel, that way you get nice and strong bands. Make sure you do a plasmid-only control when you run your gel in case it overlaps with your amplified fragments. One more idea is to double check the Tm's of your primers. Use an annealing temp about 5 deg C below the Tm's.

It's standard protocol for oligo synthesis companies to send primers in a lyophilized form to prevent degradation during shipping and so that the buyer can resuspend samples at whatever concentration they'd like. So no the company wouldn't send a sample in 10ul.

yesterday, i got these plasmids and primers from a company,
i ran them, only #3 and #5 plasmid/primer sets worked
today, i re-ran everything, only #2 and #4 plasmid/primer sets worked
(no, i didn't label them incorrectly becuz the sizes do correspond to the expected sizes)

what could happen here?
the only explanation i can come up with is that i didn't get enough plasmid at all (10pg per uL) and only about 10 uL in total for each plasmid, for #3 and #5 today, i had to spin several times just to get enough for my reaction

what else could have happened?

Several things may have occurred:

1. Your primers might be GC rich in content and preferentially anneal to each other as opposed to your plasmid target site. If that happens your PCR rxn will primarily yield primer dimers or no rxn at all. You'll be able to see the primer dimers on an EtBr stained gel under UV and it'll run at very low MW (<100bp.) on a 2% agarose gel.
2. During PCR it's standard protocol to denature ~96C for 1-2 minutes prior to starting annealing, extension, denaturation cycles. Make sure you do this step & that annealing temp is optimal. If there's too much primer dimers, try increasing the annealing temp a few degrees.
3. Run + & - controls i.e., samples with & w/o template DNA so that you know your primers are fine.
4. In using too much template DNA, primers will not have sufficient time to "find" their intended target sites during annealing. To correct for this, use serial dilutions of your template DNA. (Ex: 1/25, 1/50, 1/100, 1/200, 1/500).
5. Lowering the annealing temp will increase non-specific hybridization of your primers during the annealing step. A general rule of thumb is if you want to increase hybridization specificity between primer and target sites, increase the annealing temp.
6. Some strains of bacteria are recombinant + and can "spit" out the plasmid. Therefore, during plasmid DNA isolation, your yield is practically zero. Check and make sure your bacterial strain is recombinant -.
7. Make sure all your buffers are good and don't contain DNAses. Not only ones used for PCR but solutions used to resuspend oligos and DNA. Make sure the PCR buffer contains Mg2+ at the proper concentration. If I recall correctly, optimal [Mg2+] = 1.5mM for PCR.
8. Your dNTPs might have got bad. dNTPs are unstable and degraded faster at higher temperature. When not used, dNTPs should always be stored at -20C or lower temps. If dNTPs are suspect, make a fresh batch.

That's all I can think of from memory.

Read up on the trouble shooting sections for PCR either in "Current Protocols for Molecular Biology" (Wiley Press) or "Molecular Cloning" (Cold Spring Harbor Press) or whatever PCR book readily available to you.

Edit: Here's a "trick" I learned from a postdoc on how to do PCR without isolating plasmid DNA. If you have the agar plate with single bacteria colonies containing the plasmid, take a sterile toothpick using it to pick up a few bacteria on the toothpick end. A quick jab will do. Dip the toothpick end into a PCR mix containing all the components for rxn, except template DNA. Run PCR cylcles.

 

[Tropicana100]

It's standard protocol for oligo synthesis companies to send primers in a lyophilized form to prevent degradation during shipping and so that the buyer can resuspend samples at whatever concentration they'd like. So no the company wouldn't send a sample in 10ul.

the OP says he has 10ul of plasmid, not primer.

 

[coralfangs]

stupid question

is it possible that i made some errors pipetting?
that's the only thing i can think of
but pcr is supposed to quite robust
and i pipette everything gently up/down like usual, and i've done a lot of pcrs b4

 

[Tropicana100]

stupid question

is it possible that i made some errors pipetting?
that's the only thing i can think of
but pcr is supposed to quite robust
and i pipette everything gently up/down like usual, and i've done a lot of pcrs b4

there's only so much we can suggest with the limited info you provide - what is your plasmid (its size?, is it GC rich?), how big is your target, Tm's, what kind if pcr reagents/polymerase you use, what primer concentration, what are your cycling parameters, etc. etc? it's virtually impossible to diagnose your problem. i say just retry with freshly retransformed plasmid DNA and see what happens.

 

[Foghorn]

stupid question

is it possible that i made some errors pipetting?
that's the only thing i can think of
but pcr is supposed to quite robust
and i pipette everything gently up/down like usual, and i've done a lot of pcrs b4

It doesn't matter if the PCR is robust. If one of the components, be it template, primers, dNTPs, Taq, buffers etc., is bad the rxn won't work. That's why running + and - controls helps in figuring out if all the components are working properly.

And pipetting is not a problem unless the pipettors are way off in calibration or you do it to such an extent that it produces bubbles in your pcr reaction tube. There's enough surface tension in the bubbles to disrupt protein quaternary and tertiary structure, in this case Taq Polymerase or whatever high temp DNA polymerase you are using.

Edit: You don't even have to pipet besides adding the components in a pcr reaction tube because the heating cycles will mix the components by thermal convection.

 

[PseudomonasPAO1]

As previous people stated, first thing is to transform that plasmid into DH5a. Then you can troubleshoot without getting panicked! As for the PCR reaction, I almost always ran a gradient PCR when working with new DNA/primers. I would generally go from -5 to +5 around the Tm. Try that and see what happens.

Good luck!

 

[Aynsl156]

wait, your template is at 10pg/ul? ****, that's really little DNA. We always use about 20ng of 2-3 kb templates, and that's always worked for us. If you want a more robust reaction, I would transform the plasmids and do a mini, and then PCR from that. You could try using a hot-start polymerase, since I find that those a more robust. But make sure to heat it at 95.0*C for fifteen minutes before you start cycling.

How many cycles are you doing? Do your bands look streaky? Are you leaving your reaction at RT before adding loading buffer? I find that DNA degradation happens when I a) run too many cycles of a reaction b) leave my made-up PCR reaction at RT prior to starting the thermocycler or c) leave my finished PCR at RT for a while before adding loading buffer.

hope this helps

 

[Beeef]

It's standard protocol for oligo synthesis companies to send primers in a lyophilized form to prevent degradation during shipping and so that the buyer can resuspend samples at whatever concentration they'd like. So no the company wouldn't send a sample in 10ul.



Several things may have occurred:

1. Your primers might be GC rich in content and preferentially anneal to each other as opposed to your plasmid target site. If that happens your PCR rxn will primarily yield primer dimers or no rxn at all. You'll be able to see the primer dimers on an EtBr stained gel under UV and it'll run at very low MW (<100bp.) on a 2% agarose gel.
2. During PCR it's standard protocol to denature ~96C for 1-2 minutes prior to starting annealing, extension, denaturation cycles. Make sure you do this step & that annealing temp is optimal. If there's too much primer dimers, try increasing the annealing temp a few degrees.
3. Run + & - controls i.e., samples with & w/o template DNA so that you know your primers are fine.
4. In using too much template DNA, primers will not have sufficient time to "find" their intended target sites during annealing. To correct for this, use serial dilutions of your template DNA. (Ex: 1/25, 1/50, 1/100, 1/200, 1/500).
5. Lowering the annealing temp will increase non-specific hybridization of your primers during the annealing step. A general rule of thumb is if you want to increase hybridization specificity between primer and target sites, increase the annealing temp.
6. Some strains of bacteria are recombinant + and can "spit" out the plasmid. Therefore, during plasmid DNA isolation, your yield is practically zero. Check and make sure your bacterial strain is recombinant -.
7. Make sure all your buffers are good and don't contain DNAses. Not only ones used for PCR but solutions used to resuspend oligos and DNA. Make sure the PCR buffer contains Mg2+ at the proper concentration. If I recall correctly, optimal [Mg2+] = 1.5mM for PCR.
8. Your dNTPs might have got bad. dNTPs are unstable and degraded faster at higher temperature. When not used, dNTPs should always be stored at -20C or lower temps. If dNTPs are suspect, make a fresh batch.

That's all I can think of from memory.

Read up on the trouble shooting sections for PCR either in "Current Protocols for Molecular Biology" (Wiley Press) or "Molecular Cloning" (Cold Spring Harbor Press) or whatever PCR book readily available to you.

Edit: Here's a "trick" I learned from a postdoc on how to do PCR without isolating plasmid DNA. If you have the agar plate with single bacteria colonies containing the plasmid, take a sterile toothpick using it to pick up a few bacteria on the toothpick end. A quick jab will do. Dip the toothpick end into a PCR mix containing all the components for rxn, except template DNA. Run PCR cylcles.

😱