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

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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 10-06-09 well 6 Adineta 100+ 2 H20 4.75 28.5
CC A1 Adineta subs 100+ 5 10X Buffer 10X 1 6
WH1 B3 Adineta 20+ 7 dye 10X 1 6
WH1 B5 Adineta 20+ 8 dNTP 10mM 0.2 1.2
WH1 B6 Adineta 50+ 9 primer mix 5uM 1 6
Taq 5U/uM 0.05 0.3
EXTRACTION DNA variable 2
10-20 individuals Samples 5, 7, 8, 9 TOTAL 10 48
Cocktail per well = 8.0ul
InstaGene Matrix 200ul per sample
56C 30 min Primer HIS F1+R1
99.9C 8min 5475 F + 8047 R
load 2.0ul 1kb ladder and 2.0ul sample 1% gel PCR 95C 3 min
Sample #2 Positive Control 95C 10 sec
SAMPLE Gel Well 60C 2 min
HIS F1+R1 Sample 2 1 35 cycles
HIS F1+R1 Sample 5 2
HIS F1+R1 Sample 7 3
HIS F1+R1 Sample 8 4
HIS F1+R1 Sample 9 5
5475 F + 8047 R Sample 2 6
5475 F + 8047 R Sample 5 7
5475 F + 8047 R Sample 7 8
5476 F + 8047 R Sample 8 9
5476 F + 8047 R Sample 9 10
07dec09 gel text.jpg

Adineta Amp Primer 7-12

Plate Well Genus 12/2/2009 Count Sample Extract ' Reagents Concentration 1X 15X
Adenita 23Nov09 B3 Adineta 30+ 1 H20 4.75 71.25
AV 10-06-09 well 6 Adineta 100+ 2 10X Buffer 10X 1 15
Boston A1 Adineta 10+ 3 dye 10X 1 15
Boston A4 Adineta 50+ 4 dNTP 10mM 0.2 3
CC A1 Adineta subs 100+ 5 primer mix 5uM 1 15
CC A3 Adineta subs 100+ 6 Taq 5U/uM 0.05 0.75
WH1 B3 Adineta 20+ 7 use 12/7 DNA variable 2
WH1 B5 Adineta 20+ 8 use 12/7 TOTAL 10 120
WH1 B6 Adineta 50+ 9 use 12/7 Cocktail per well = 8.0ul
WH1 C4 Adineta 30+ 10
WH2 A6 Adineta 10+ 11 On Gels
WH3 A5 Adineta 20+ 12 Primers AV10130 #1-13
WH3 C2 Adineta 7 13 AV10123 #14-26
AV10121 #27-39
EXTRACTION AV10104 #40-52
InstaGene Matrix 200ul per sample AV10109 #53-65
56C 30 min AV10099 #66-78
99.9C 8min
PCR 95C 3 min
95C 10 sec
12/2/2009 5-10 individuals 12/7/2009 10-20 individuals 60C 2 min
35 cycles
load 2.0ul log ladder and 2.0ul sample 1% gel
Sample #2 Positive Control
09dec09 redo 08dec09 gel 1-19 text.jpg
08dec09 20-38 cropped.jpg
08dec09 39-60 text.jpg
08dec09 61-78 text.jpg