PCR Question

[Mega]

Hi everybody.


I'd just like to know if you could possibly do a PCR from two sources at a time  using three primers?



So e.g.:

Usually if you want to have a GFP-KanR construct, you would use four primers, with the primers between the two genes of interest having the same restriction site. Those would then be ligated later.

What if you took just one primer which contains the end of GFP + a short linker + beginning of KanR   and of course one GPF starting primer and one KanR end primer? Would that still work?

Additionally, the sources were two different plasmids. Could that possibly work, to get one construct in one step (without the need of restriction digestion and re-ligating)

May it be a problem that the ends of primers can have a  false sequence?

[Nathan McCorkle]

except the middle primer you envision is going toward the GFP gene,
whereas it needs to go toward the KanR gene... remember 5' to 3' is
the way the enzymes read/elongate DNA

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--
-Nathan

[Nathan McCorkle]

it's possible that the newly created strand from the first two primers
would then actually prime the KanR area as the forward primer... seems
like you could easily simulate the outcome of this

--
-Nathan

[Andreas Sturm]

But the middle primer has one problem, doesn't it?

I remember a professor adding TATATAT to each end of the primer, because he said the end may not be correctly synthesized??

[Andreas Sturm]

So you mean, the problem is that the GFP then is not necissarily amplified?

[Josiah Zayner]

People add bases to end of primers because restriction enzymes tend to bind on both sides of the cleavage site.

[Nathan McCorkle]

you need a forward AND a reverse primer. If you encode the forward
primer for the KanR in the reverse primer of the GFP, then the forward
primer won't 'come alive' until the GFP gene has gone through PCR
twice. Once for the reverse GFP primer to extend, and once for the
forward GFP primer to extend ON THE NEW STRAND FORMED BY THE REVERSE
PRIMER. This resulting strand will be the correct sense to act as a
the forward primer for KanR.

I guess you'd only want a very small amount of the middle primer, to
avoid too many GFP-only amplifications.

I don't understand why your prof would use TATATATA at the end of a
primer unless they wanted to add that sequence to their amplicon, or
they hoped it would aide priming if the primer didn't match perfectly.

--
-Nathan

[Nathan McCorkle]

On Mon, Jan 7, 2013 at 1:17 PM, Josiah Zayner <josiah...@gmail.com> wrote:
> People add bases to end of primers because restriction enzymes tend to bind
> on both sides of the cleavage site.
>

ahh sure, that's reasonable.

--
-Nathan

[Mega]

So explicitly said: the enzyme needs something to grab next to the binding site?

[Josiah Zayner]

Correct.

See: http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/cleavage_olignucleotides.asp#.UOxCDKzNlks

[Nathan McCorkle]

That's a restriction enzyme not PCR polymerase

On Jan 7, 2013 11:57 PM, "Mega" <masters...@gmail.com> wrote:

So  explicitly  said:   the enzyme needs something to grab next to the binding site?

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[Josiah Zayner]

I believe that is what we were talking about. However, forgive me if the conversation was going somewhere else.

[Andreas Sturm]

Josiah, that was also helpful, thanks.

So basically, a primer should have a certain lenght (it says as a general rule 6 bp), so that the restriction encyme doesn't cut unsymetrically.



As for the double-construct, it may go wrong due to only the GFP being multiplied first... So basically it's better to make 2 PCRS and ligate together?

[Andreas Sturm]

Well, what might be feasible:

Do some PCR cycles, some of it will be only- GFP and some GFP-KanR.


Then do another PCR using this as a template.  Just add forward and reverse primers, no middle primer ->  it will only amplify the construct.





I am also quite sure that this was also what you ment, Nathan. But your approach was in one step.

[Jeswin]

On Tue, Jan 8, 2013 at 2:36 PM, Andreas Sturm <masters...@gmail.com> wrote:
> Well, what might be feasible:
>
> Do some PCR cycles, some of it will be only- GFP and some GFP-KanR.
>
>
> Then do another PCR using this as a template. Just add forward and reverse
> primers, no middle primer -> it will only amplify the construct.

>Hmm, let me see if I understand this. So you have 2 pcr products and you want to combine them, sort of like in pcr mutagenesis reactions? I think that will work if you use the right primers.

[Eugen Leitl]

On Tue, Jan 08, 2013 at 01:15:29PM -0600, Josiah Zayner wrote:
> I believe that is what we were talking about. However, forgive me if the
> conversation was going somewhere else.

Please do not top-post and please trim your replies. Message
unchanged below for illustration.

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______________________________________________________________
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[Eugen Leitl]

On Tue, Jan 08, 2013 at 11:10:06AM -0800, Nathan McCorkle wrote:
> That's a restriction enzyme not PCR polymerase

Please do not top-post and please trim your replies. Message
unchanged below for illustration.

[Nathan McCorkle]

Yes if you limit the middle primer to say 1/10th of the two outer
primers I think this would work in a single reaction. You don't need
to break it up into two reactions unless you plan to gel purify the
amplicon first.

Polymerase and ligase also actually need some space to sit down on the
DNA strand, I can't remember if it was a nuclease, polymerase or
ligase paper that I read using testing lots of small lengths down to 6
or 8bp.... I couldn't find that paper (was told to organize my PDFs
with Zotero, so I'll try doing that now) but I found this one showing
Taq polymerase working with 14bp primers and a isothermal polymerase
working on a 10bp primer
http://handelsmanlab.sites.yale.edu/sites/default/files/MiniprimerPCR.pdf

-Nathan

[Andreas Sturm]

Ah ok.

On Tue, Jan 8, 2013 at 9:38 PM, Nathan McCorkle <nmz...@gmail.com> wrote:

Yes if you limit the middle primer to say 1/10th of the two outer
primers I think this would work in a single reaction. You don't need
to break it up into two reactions unless you plan to gel purify the
amplicon first.

That's good, because the forward primer should have 130 bp of a promoting region added ;)


Can you do PCR with such big fat primers? (In total a little less than 160 bp for the first primer)
 

[Nathan McCorkle]

Sure but an optimal PCR reaction will have the melting temps be quite
close to each other (within a degree or so), a long 150bp+ DNA is
gonna have a much higher melting temp than a 30bp primer unless the
long one had a higher AT:GC ratio than the shorter one (two equal
length DNAs GCGCGCGC.... and ATATATAT.... the GC one will have a
higher melting temp because GC binds with 3 H bonds not 2)

--
-Nathan

[Cathal Garvey]

Actually, most restriction enzymes won't cut at all unless they can cut both strands. Because they operate as a dimer, they must "meet" around the active site and either activate each other, or work together to change the DNA shape prior to cutting. So, if an enzyme monomer can't "sit" on one side of the DNA, it can't form an active dimer, and won't cut.

If memory serves, *most* enzymes will work with 3bp to either side of the target site, although efficiency may suffer and it's not an absolute rule. For the cost of 6 extra BP, it's better to just establish a general rule of thumb and work with it.

I'm actually planning to do a partial assembly, partial amplification PCR at the moment, so this is relevant to me. Overlap PCRs should work as you initially laid out in a no-cut, single step reaction, although the efficiency will vary from PCR to PCR, as usual. So sometimes, it's a one-step-wonder and you just get the desired product, and other times it'll fail entirely.

At least if you do it the way you initially planned, the whole reaction will likely fail, provided you add only a small quantity of the "middle" primer. Here's some PCR ASCII (that'll probably fail to render in most mail clients, so copy/paste to gedit or notepad):

F1: ---->
T1: ==============
R1:          <-------
T2:               ===============
R2:                         <----

Legend:
F1 = Forward primer 1
R1 = Reverse primer 1
R2 = Reverse primer 2
T1 = Template 1
T2 = Template 2

Rough protocol:
Add normal amounts of F1 and R2
Add maybe 1/10 amount R1
Add normal template amounts T1, T2
Run as usual with extension step tailored for full T1T2 product.

Expected process:
First ~5 rounds, T1+R1 amplified.
Round 3 onwards: Amplification product becomes forward "primer" for T2 template.
Primer R1 becomes depleted early, preventing wastage of NTP etc. on T1-only product.
Hopefully, reaction proceeds to amplify T1T2 using F1 and R2.

Has anyone else tried similar PCRs? I haven't! :)

www.indiebiotech.com
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joindiaspora.com/u/cathalgarvey
PGP Public Key: http://bit.ly/CathalGKey

[Mega]

Hi,

reviving this thread for one more question ;)


I did a PCR of a 9-10 kbp plasmid and the product should have been some 6300 bp long.

Now on this gel you see two bands (+primers), one at 9kbp and one at 6 kbp. May this be template plasmid + product? Or is this template (circular plasmid) + template (supercoiled) ?

Best,
Andreas

[Andreas Sturm]

Here some pics...

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[Nathan McCorkle]

Which column are you questioning? You could have done a better
photoshop/GIMP/MsPaint job! Usually labels go above or below lanes,
not on the side with pointing lines.

See the first page here:
http://people.rit.edu/rhrsbi/GEPages/LabManualPDF5ed/15%20electrophoresis.pdf

basically a way to be sure is by cutting the plasmid initially.

But I think if you used the same amount of plasmid in the PCR reaction
as you did in the control lane (plasmid only) you could tell if the
PCR worked simply by the difference in band brightness!

I'm actually not sure what any of your lanes are, which is the ladder,
which ladder is it? Did you use a ladder (prepared standard size
fragments)?

>
> Best,
> Andreas
>
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--
-Nathan

[Andreas Sturm]

Oh, sorry I didn't write that down... On the left side there is Lambda Hind DNA ladder and then Benchtop 100 bp ladder (the biggest 1500 bp)  Then comes another plasmid which is not of interest.

The last four smaples are the samples of interest

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[Andreas Sturm]

Don't they look a bit like primer dimers?

[Nathan McCorkle]

On Sat, Jul 13, 2013 at 12:40 AM, Nathan McCorkle <nmz...@gmail.com> wrote:
> you could tell if the
> PCR worked simply by the difference in band brightness!

Same idea for primer-dimers, but you didn't do a control lane for
plasmid-only and primers-only...

--
-Nathan

[shreyans chordia]

If u want to avoid the 9kb band, try to use less PCR template as possible.

[Jeswin]

On Sunday, July 14, 2013 5:59:51 PM, shreyans chordia wrote:
> If u want to avoid the 9kb band, try to use less PCR template as possible.
>

I was going to say that too. Usually, you never see the plasmid
template band after pcr. What's your pcr enzyme and cycle conditions?
I'm suprised at the size. I would think it might have been better to
create multiple fragments and pcr combine them. Most likely will be
less prone to errors.

[Mega]

Errors shouldn't be a problem as this is Phusion proofreading polymerase,,,

Cycle conditions according to https://www.neb.com/protocols/1/01/01/pcr-protocol-m0530

Initial Denaturation	98°C	30 seconds
25-35 Cycles	98°C 45-72°C 72°C	10 seconds 10-30 seconds 30 seconds per kb. 6300 bp -> 3 mins
Final Extension	72°C	8  minutes
Hold	4-10°C

32 cycles.

[Mega]

Forgot to mention:  temperature gradient 52-60°C
and 30 seconds (always the longer time was used)