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

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Contents

Adineta Primers 25-30 ReAmp

Plate Well Genus 12/2/2009 Count Sample Extract ' Reagents Concentration 1X 14X
AV 10-06-09well 6Adineta100+2H204.7566.5
BostonA1Adineta10+310X Buffer10X114
BostonA4Adineta50+4dye10X114
CCA1Adinetasubs 100+5dNTP10mM0.22.8
CCA3Adinetasubs 100+6primer mix5uM114
WH1B3Adineta20+7use 12/7Taq5U/uM0.050.7
WH1B5Adineta20+8use 12/7DNA variable2
WH1B6Adineta50+ 9use 12/7TOTAL10112
WH1C4Adineta30+10Cocktail per well = 8.0ul
WH2A6Adineta10+11
WH3A5Adineta20+12On Gels
WH3C2Adineta713PrimersAV10034#1-12
AV10028#13-24
AV10027#25-36
EXTRACTIONAV10025#37-48
InstaGene Matrix 200ul per sampleAV10016#49-60
56C30 minAV10011#61-72
99.9C8min
PCR 95C3 min
95C10 sec
12/2/20095-10 individuals12/7/200910-20 individuals60C2 min
35 cycles
load 2.0ul 2-log ladder and 2.0ul sample 1% gel
Sample #2 Positive Control

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.

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