PCR end weight

[Koeng]

Hello all!

I am using the gibson assembly and it requires a certain amount of dna, but I don't know how much dna my PCR reaction will create! Is there any equations, or recommendations on how much I should use if in my reaction I used .2 µM of primer in each reaction?

[Avery louie]

I dont know of any way to know how much you are going to create- normally you have to quantify it with a UV spectrophotometer.

--A

On Wed, Mar 13, 2013 at 11:48 AM, Koeng <koen...@gmail.com> wrote:

Hello all!

I am using the gibson assembly and it requires a certain amount of dna, but I don't know how much dna my PCR reaction will create! Is there any equations, or recommendations on how much I should use if in my reaction I used .2 µM of primer in each reaction?

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[Dakota Hamill]

You can also estimate the amount by comparing band intensity to a ladder with known concentrations of DNA.

https://www.neb.com/products/N3232-1-kb-DNA-Ladder

[Nathan McCorkle]

To do the calculation you desire, we'd need to know the length of the
amplicon (what you're trying to PCR).

You can estimate grams out using the average mass of a nucleotide, and
assuming your PCR reaction goes until completion (you use all the
primers). You can do it based on number of cycles and starting DNA
present, but this way is much easier.

If you go until completion, you can assume your 0.2 micromoles of
primer have simply turned into 0.1 or 0.2 micromoles of full-length
amplicon (depending on if the 0.2 micromoles was one only or both of
your primers total). The important part is that since 2 moles of
primer go to 1 mole of amplicon, that you do the math right. (Left
primer and Right primer join in the middle, going from 2 molecules to
1 molecule).

So if I assume you added 0.2 micromoles of Forward primer and the
amplicon is 500bp long, at completion there will be 0.2 micromoles of
amplicon. A quick googling shows the average for dsDNA "1 pmol of 1000
bp DNA = 0,66 µg" so:
(.66uG / 1000bp) * picomoles_per_micromole * micromoles_present * base_pairs
(.66/1000) * 1000000 * .2 * 500 = 66000 micrograms

if it was done in 50uL reaction, that's 1320 uG/uL


Again, this is assuming the PCR goes to completion (no remaining
primer) and no mispriming happens (this will contribute to the final
mass, but it's noise and you won't know exactly what the length is) or
polymerase falling off and not completing an amplicon completely (this
is why you generally have the last PCR cycle much longer, to allow it
to play catch-up)

--
-Nathan

[Koeng]

I did some calculations myself using some of yours (I had to go back and track everything I did) I found I had around 3.5 pmols per uL. (if everything went correctly)

So, for the gibson assembly, I had 2 different reactions. One was with about 10 pmol and the other was with about .5 pmol, of each fragment.

Now here comes the tricky part. I need to do a  transformation today and I plan on using this protocol

https://github.com/cathalgarvey/biohacking-protocols/blob/master/E.coli%20Transformation%20With%20PEG%2BMgSO4.md

How much of the DNA should I use? I need to save some for verification, and some I need to use for transforming the Halobacteria! It is currently in a 20µl solution, so what do you guys think I should I dilute it to? Thanks for the help!

[Dakota Hamill]

Can archaeic bacteria be transformed the same way E. Coli can?  Don't they have an extra cell membrane and need crazy salt concentrations just to live?  

Or are you going to grow up your plasmid in E. Coli then miniprep it ?

What exactly are you trying to assemble, or is it top secret?

[Koeng]

I am trying to assemble a Halobacteria vector for the model organism Halobacteria NRC-1, and not top secret, I want to give the plasmid out for free once I finish xD

I am thinking about doing a miniprep but I have to do a transformation today or else I fear my experiment will not have results in time for my science fair...

Yes they do need crazy salt concentrations, it gets very annoying because the when I pour the agar, everywhere it touches turns into salt :/

I do not believe they have an extra membrane, considering E coli has a protein layer too I think the PEG will still work, however I need to test CaCl method as well to get the best results.

Thanks

On Wednesday, March 13, 2013 8:48:45 AM UTC-7, Koeng wrote:

[Dakota Hamill]

May the force be with you.