High altitude PCR 2: preliminary data!

As promised on Friday, here is some initial data for this idea.  I still don't know if it truly is an effect of altitude or just denature temperature, but here is what I did.  I set up 8 identical reactions (10uL each) to amplify an 800bp product from pUC19 and ran them under three different cycling conditions on the same cycler on the same day (barometer = 30.43 in Hg, corrected boiling point = 93.38C).  I note that there was a lot of condensation present on the upper portion of the wells following Cycle 1, but much less after Cycle 2 or Cycle 3.

Cycle 1
1) 95C 1 min
2) 95C 30s
3) 55C 30s
4) 72C 45s
5) goto 2, 29X

Cycle 2
1) 90C 1 min
2) 90C 30s
3) 55C 30s
4) 72C 45s
5) goto 2, 29X

Cycle 3
1) 90C 1 min
2) 80C 30s
3) 90C 30s
4) 55C 30s
5) 72C 45s
6) goto 2, 29X

I ran 2uL each product on a gel:

Everything looks about the same, but you might notice there are more large (greater than 800bp) non-specific products in the 95C denature and the 80C/90C denature programs than the 90C denature alone.  I think this is partially because 30 cycles may have been too many for this amplicon.  I hope to rerun this analysis with fewer cycles another day.

That said, significance was achieved (ANOVA p = 0.0104)!  Again, I think this is largely due to the added non-specific products.  Here is a nice bar graph showing the results with Tukey's HSD separations denoted. The concentrations were obtained using OD260 method following ethanol precipitation of all products.  Each sample was resuspended in 50uL 1X TE.

What can I take away right now?  90C is adequate for denature of a simple bit of DNA such as pUC19 and I know it avoids boiling my sample at this altitude.  Less condensation appears on the upper portion of the tube walls under a 90C denature, presumably preventing concentration of reagents which can lead to production of non-specific bands.  The effect, regardless of its cause, results in a significant difference in total PCR yield, but presumably more of the product in the 90C denature is the product I want while the other cycles produce unwanted by-products.

Where to go next?  Repeat with fewer cycles, possibly use a qPCR to guide the appropriate number of cycles, measure with a pipet the volume of reaction that remains at the bottom of the well following each cycle next time.  For now, reduce denature step to 90C.  When this particular reaction works a bit better, test it again at different altitudes.  Also maybe test a more complex target.

At the very top of Snowbowl (~11,500ft, if they will loan me an outlet for the day), the boiling point would be 89.2C on the same day, thus boiling all of my reactions.  In the Verde valley (~3,500ft), the boiling point would be 96.9C thus eliminating all boiling for all reactions.

A colleague told me this weekend his new motto is "free Tibet (to do PCR)!"