PCR problemo, and advice?

[Koeng]

Hey guys,

I am doing one of my first own pcrs with my machine and tubes, when the sudden realization that the pcr machine filled .5 (I think) ml when I had .2 ml tubes! Does anyone think that it will still work? Anyway, I am on a strict time table, so I am going to PCR it and check the results on Sunday (my UV light doesn't work for my electrophoresis, so much for being cheap and getting storebought blacklight when i lost my good UV light). So who wants to give a guess if it will work or not?

[Avery louie]

"the pcr machine filled .5 (I think)"

I am confused by this.

What kind of machine?

--A

On Tue, Mar 12, 2013 at 11:42 PM, Koeng <koen...@gmail.com> wrote:

Hey guys,

I am doing one of my first own pcrs with my machine and tubes, when the sudden realization that the pcr machine filled .5 (I think) ml when I had .2 ml tubes! Does anyone think that it will still work? Anyway, I am on a strict time table, so I am going to PCR it and check the results on Sunday (my UV light doesn't work for my electrophoresis, so much for being cheap and getting storebought blacklight when i lost my good UV light). So who wants to give a guess if it will work or not?

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[Koeng]

it is filled with .5 ml tubes, since the others I have have a lot of of space around them when I put them in, and my 1.6 centrifuge tubes would not fit.

Coy 110S TempCycler II Thermal Cycler is the exact machines name.

[Avery louie]

People do pcr in 0.5 ml tubes...My money is on it working.

--A

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[Koeng]

K, thats what I thought when I bought the .2 ml tubes :)

Just making sure with you guys because I spent so much on this science fair project (and I am planning on giving out the result to the DIY bio community, an open source halobacterium NRC-1 vector, compatible with biobricks and all :D )

[Josh Perfetto]

The key for this working is to ensure that the business portion of the PCR tube (the portion containing the reaction volume and a bit above it) makes excellent contact with the PCR machine block. If it fits snug that's great, if there's an air gap the reaction temperature control will be way off. If you have a gap, then in a pinch you might make this work by putting mineral oil (or cooking oil) between the heat block and tube so as to fill in the gap. And you may need to extend your step hold times compared to a 200 ul tube, as it will still take longer for your reaction volume to reach the block hold temperature.

-Josh

On Tue, Mar 12, 2013 at 8:42 PM, Koeng <koen...@gmail.com> wrote:

Hey guys,

I am doing one of my first own pcrs with my machine and tubes, when the sudden realization that the pcr machine filled .5 (I think) ml when I had .2 ml tubes! Does anyone think that it will still work? Anyway, I am on a strict time table, so I am going to PCR it and check the results on Sunday (my UV light doesn't work for my electrophoresis, so much for being cheap and getting storebought blacklight when i lost my good UV light). So who wants to give a guess if it will work or not?

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[Avery louie]

Josh makes a good point- I have had PCR go bad that way (hence my need for .2ml tubes). I assumed that the .5 ml tube fit ok.

--A

[Avery louie]

This paper seems relevant here- pcr tube choice is important.  link

[Koeng]

Thanks, I think I have to retry the pcr reaction because I do not have a heated lid, and forgot to put oil on it... almost everything was evaporated....

So this time I put cooking oil on the lid and in the wells, and increase the hold times.

Koeng

On Tuesday, March 12, 2013 8:42:28 PM UTC-7, Koeng wrote:

[Dakota Hamill]

If you give a little more information on the concentrations and volumes of the PCR reaction, as well as the expected product size and program times we might be able to help a little more.  Also, you can pick up mineral oil for $2 from a local pharmacy, it's also known as "baby oil" but baby oil just has fragrances added, so non-scented baby oil works too.  Just check the main ingridients

[Josh Perfetto]

Just one thing - when I said you could use cooking oil I meant that only for the area between the well and the tube. If the oil in the tube is not DNAse free it may mess up your PCR. Let us know if it works though.

-Josh

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