User:Alexander S Mikheyev/Notebook/rotifer alien genes/2009/12/14

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Adineta Primers 25-30 ReAmp

Plate Well Genus 12/2/2009 Count Sample Extract ' Reagents Concentration 1X 14X
AV 10-06-09 well 6 Adineta 100+ 2 H20 4.75 66.5
Boston A1 Adineta 10+ 3 10X Buffer 10X 1 14
Boston A4 Adineta 50+ 4 dye 10X 1 14
CC A1 Adineta subs 100+ 5 dNTP 10mM 0.2 2.8
CC A3 Adineta subs 100+ 6 primer mix 5uM 1 14
WH1 B3 Adineta 20+ 7 use 12/7 Taq 5U/uM 0.05 0.7
WH1 B5 Adineta 20+ 8 use 12/7 DNA variable 2
WH1 B6 Adineta 50+ 9 use 12/7 TOTAL 10 112
WH1 C4 Adineta 30+ 10 Cocktail per well = 8.0ul
WH2 A6 Adineta 10+ 11
WH3 A5 Adineta 20+ 12 On Gels
WH3 C2 Adineta 7 13 Primers AV10034 #1-12
AV10028 #13-24
AV10027 #25-36
EXTRACTION AV10025 #37-48
InstaGene Matrix 200ul per sample AV10016 #49-60
56C 30 min AV10011 #61-72
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 2-log ladder and 2.0ul sample 1% gel
Sample #2 Positive Control
14dec09 pimer25-30 1-38 text.jpg
14dec09 pimer25-30 39-60 text.jpg
14dec09 pimer25-30 61-72 text.jpg

11Dec09 Cycle Sequencing Post-reaction Clean Up & Resuspension

http://jbpc.mbl.edu/SeqFacility/Pages/Precipitation/precipprotocol.html

Steps 4 - 9 should be carried out without delay to prevent loss of pellets. DNA in formamide will degrade even if kept frozen. Reactions should be precipitated within 24 hours. After resuspension they should be sequenced within 3-5 days.

Supplies: Isopropanol 75 % and 70%, pipette tips, reservoir tray, paper towels, Costar foil sealer, HiDi formamide

Spin plate briefly.

Add 30µl 75% Isopropanol, seal with Costar foil, invert a few times to mix, shake sample to bottoms of wells.

Incubate at room temp for 15 minutes.

Centrifuge at 2800 RCF or 4200 RPM for 30 minutes.

Remove foil, invert on paper towel, "swirl" inverted plate and paper towel in circular motion on benchtop until most IPA falls out. Do not bang/slap/slam/tap inverted plate or pellet could be lost. (Alternatively a short spin at 100-200 RCF can be used. It is not necessary to get every last bit of IPA out of the wells.)

Add 50µl 70% IPA, seal with foil.

Spin at 2000 RCF or 3300 RPM for 10 minutes.

Remove foil and invert onto fresh paper towels.

With plate inverted on paper towel, spin at 200 RCF or 500 RPM for 1 minute.

Air dry for 20 minutes in the dark.

Resuspend in 7µl HiDi Formamide. (Note: Add formamide to all wells, even if you do not have a full plate of samples, or you will cause damage to the 3730XL capillary array.)

Spin plate briefly.

Seal with Costar foil. Store in freezer and plan to sequence within 3 days.