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

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==Task==
==Scun22 PCRs from 7 and 10 February==
* here
* These are from the PCRs that Rulon and I demo'd to the students
* here
:* In both cases, used Scun22 primers, run on both Scoc and Scun DNA.
** in here
:* We used the same PCR sheet, but Rulon put his samples in A12-H12 of his PCR plate. Same reaction volumes used.
# numbered
:* Rulon PCR'd 7 Feb 2011; Tara PCR'd 10 Feb 2011
# again
:* [[Image:20110221 PCR.jpg|800 px]]




===Subsection===
==Scun22 Agarose Gel==
* here
* 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 MgCl<sub>2</sub>, 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==
* [http://dps.sdsu.edu/defendrive.htm 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
* [http://www.dfg.ca.gov/wildlife/nongame/research_permit 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




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Revision as of 22:35, 4 March 2011

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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
  • ----
  • 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
  • -
  • ---
  • 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
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
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