User:Tara K. Luckau/Notebook/Team ConGen/2011/02/21

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Scun22 PCRs from 7 and 10 February

  • These are from the PCRs that Rulon and I demo'd to the students
  • In both cases, used Scun22 primers, run on both Scoc and Scun DNA.
  • We used the same PCR sheet, but Rulon put his samples in A12-H12 of his PCR plate. Same reaction volumes used.
  • Rulon PCR'd 7 Feb 2011; Tara PCR'd 10 Feb 2011

Scun22 Agarose Gel

  • Pour small Gel: 50 mL 1x TAE + 1 g agarose + 5 µL GelRed
  • Load: 5µL Orange/Blue loading dye + 2 µL PCR product
  • This is the first time I'm trying the Orange/Blue Loading Dye (Orange G and Xylene Cyanol FF), stolen from Joe Newsome's excess stock
  • Run: 80V, 40 min
  • ----
  • Image:20110221a_Full.TIF
  • oops, can't see product because I used the 6x loading dye like an idiot
  • did dye:DNA 1:5 instead of DNA:dye 1:5

Scun22 Agarose Gel Corrected

  • Pour small Gel: 50 mL 1x TAE + 1 g agarose + 5 µL GelRed
  • Load: 1µL Orange/Blue loading dye + 5 µL PCR product
  • Using the Orange/Blue Loading Dye (Orange G + Xylene Cyanol FF + Bromophenol Blue), stolen from Joe Newsome's excess stock
  • Run: 90V, 20 min
  • -
  • Image:20110221b_Full.TIF
  • ---
  • Image:20110221b_RightDim.TIF
  • Only two samples amplified:
  • Tara's Scoc (4th replicate) - primary band at expected ~450 bp, but with mispriming
  • Tara's Scun (1st replicate) - primary band at expected ~450 bp
  • This pattern (Scoc dirty, Scun clean) is consistent with results from 14 January

Next Steps

  • Scun22
  • Optimize by varying MgCl2, convert to standard thermal cycling profile ... full gradient PCR (temp and Buffer)
  • Scun2
  • Optimize by converting to standard thermal cycling profile
  • Try to get their temperatures to match!

Meeting with Rulon

  • completed online CSU-approved defensive driving course - send certificate of completion to DPS
  • completed two other forms ... keep with department? Ask Medora
  • CA DFG SCP - send email to Randi Logsdon requesting status update; no response, yet
  • pipet and tips
  • prefer to order GeneMate, even though it would require stocking different tips
  • Rulon gives go-ahead

Field Work

  • Sample IDs
for use in the field (to ensure each sample has a unique ID)
MMDDYY initial A, B, C ...
  • Torrey Pines sites
  • GPS coordinates
in degrees and minutes (seconds as decimals of minute)
  • Tissue sample
split into 2: one for DNA, one for isotope (no alcohol)
lizard = toe and tail
snake = scale
  • Maps
print color copies of maps, in plastic sleeve
county, site, array
  • Permits
get copies of Rulon's permits
  • Database
email Carlton for full database (so we can share our stuff with USGS at end)
  • Field Data Sheets
get copies from Rulon
data sheet, toe clip, scale clip
  • Field Kit
Pesola Scales (10g, 20g, 60g, 300g)
Rulers (stick and tape)
lab pens (2)
bunch o' baggies
alcohol wipes
ethanol tubes
hand Wipies

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