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
 * [[Image:20110221 PCR.jpg|800 px]]

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:20110221 GelSchematic.jpg|675 px]]
 * [[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:20110221 GelSchematic.jpg|655 px]]
 * [[Image:20110221b_Full.TIF]]
 * ---[[Image:20110221b GelSchematic.jpg|575 px]]
 * [[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

 * SDSU Defensive Driving
 * completed online CSU-approved defensive driving course - send certificate of completion to DPS
 * [[Image:20110218 TKLDrivingCert.jpg|200 px]]
 * 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
 * TP3-1-10
 * TP3-11-15
 * TP1-1-10


 * 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
 * forceps
 * scissors
 * QuikStop
 * alcohol wipes
 * ethanol tubes
 * hand Wipies