User:Alexander S Mikheyev/Notebook/rotifer alien genes/2009/12/07

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Contents

To do

  • Desiccate rotifers for
  • re-extract failed rotifers

Rotifer dessication

  • prepare rotifers
    • take three vials of each species to be tested
      • one as a test rehydration, one to take to Japan, one to keep in the Mark Welch lab
    • Add small pieces of filter paper to the rotifer culture, and remove most water with a pipette, leaving .3-.5 ml
    • note number of rotifers
  • prepare humidity chamber
    • The dry salt is spread about 3 mm deep in a petri dish that occupies most of the bottom of a small airtight bottle.
    • Water is added to moisten the salt -- do not add more water than is needed to make the salt look damp.
    • keep each salt for about 4 hours, then replace with the one giving a lower relative humidity.
    • keep under Potassium carbonate until dry (also for long-term storage @ rt)
    • once dry, put both vials at -80C
  • Relative humidity table
Salt RH (%)
Potassium carbonate 43.2
Sodium chloride 75.3
Potassium chloride 84.3
  • We can also use Sodium bromide (RH 57.6%) as an intermediate, if necessary

Rotifer Re-Extract Primer 35 & 36

Plate Well Genus 12/2/2009 Count Sample # ' Reagents Concentration 1X 6X
AV 10-06-09well 6Adineta100+2H204.7528.5
CCA1Adinetasubs 100+510X Buffer10X16
WH1B3Adineta20+7dye10X16
WH1B5Adineta20+8dNTP10mM0.21.2
WH1B6Adineta50+ 9primer mix5uM16
Taq5U/uM0.050.3
EXTRACTIONDNA variable2
10-20 individuals Samples 5, 7, 8, 9TOTAL1048
Cocktail per well = 8.0ul
InstaGene Matrix 200ul per sample
56C30 minPrimerHIS F1+R1
99.9C8min5475 F + 8047 R
load 2.0ul 1kb ladder and 2.0ul sample 1% gel PCR 95C3 min
Sample #2 Positive Control95C10 sec
SAMPLE Gel Well 60C2 min
HIS F1+R1 Sample 2135 cycles
HIS F1+R1 Sample 52
HIS F1+R1 Sample 73
HIS F1+R1 Sample 84
HIS F1+R1 Sample 95
5475 F + 8047 R Sample 26
5475 F + 8047 R Sample 57
5475 F + 8047 R Sample 78
5476 F + 8047 R Sample 89
5476 F + 8047 R Sample 910

Adineta Amp Primer 7-12

Plate Well Genus 12/2/2009 Count Sample Extract ' Reagents Concentration 1X 15X
Adenita 23Nov09B3Adineta30+1H204.7571.25
AV 10-06-09well 6Adineta100+210X Buffer10X115
BostonA1Adineta10+3dye10X115
BostonA4Adineta50+4dNTP10mM0.23
CCA1Adinetasubs 100+5primer mix5uM115
CCA3Adinetasubs 100+6Taq5U/uM0.050.75
WH1B3Adineta20+7use 12/7DNA variable2
WH1B5Adineta20+8use 12/7TOTAL10120
WH1B6Adineta50+ 9use 12/7Cocktail per well = 8.0ul
WH1C4Adineta30+10
WH2A6Adineta10+11On Gels
WH3A5Adineta20+12PrimersAV10130#1-13
WH3C2Adineta713AV10123#14-26
AV10121#27-39
EXTRACTIONAV10104#40-52
InstaGene Matrix 200ul per sampleAV10109#53-65
56C30 minAV10099#66-78
99.9C8min
PCR 95C3 min
95C10 sec
12/2/20095-10 individuals12/7/200910-20 individuals60C2 min
35 cycles
load 2.0ul log ladder and 2.0ul sample 1% gel
Sample #2 Positive Control
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