User:Alexander S Mikheyev/Notebook/rotifer alien genes/2010/07/26

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Alien Gene Second Cloning QiaQuick PCR Purification and Cycle Sequencing

  1. 1-32 - all fragments in right size range

http://www.qiagen.com/products/dnacleanup/gelpcrsicleanupsystems/qiaquickpcrpurificationkit.aspx#Tabs=t2

Sample ID ng/ul A260 A280 260/280 260/230 Constant
1 31.1 0.622 0.319 1.95 2.15 50
6 29.19 0.584 0.313 1.86 2.26 50
12 22.8 0.456 0.239 1.91 2.15 50
18 26.34 0.527 0.288 1.83 2.27 50
24 32.47 0.649 0.346 1.87 2.17 50
30 29.76 0.595 0.321 1.85 2.15 50


CS Reaction mixture conc. 1x 34 '
Big Dye Terminator 0.5 17
Primer (M13FWD) 5uM 0.4 13.6
DMSO 0.1 3.4
5X Seq Buffer 5X 0.4 13.6
Water 5U/ul 3.6 122.4
Template * ? 1
170 5.0ul / RXN
* Ethanol Cleaned PCR product
Cycle Seq Program
96C 10 sec
50C 5 sec
60C 4 min
x60

Alien Gene Second Cloning Post PCR Clean Up & Resuspension

1. Spin plate briefly. 2. Add 30μl 75% Isopropanol, seal with Costar foil, invert a few times to mix, shake sample to bottoms of wells. 3. Incubate at room temp for 15 minutes. 4. Centrifuge at 2800 RCF or 4200 RPM for 30 minutes. 5. 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.) 6. Add 50μl 70% IPA, seal with foil. 7. Spin at 2000 RCF or 3300 RPM for 10 minutes. 8. Remove foil and invert onto fresh paper towels. 9. With plate inverted on paper towel, spin at 200 RCF or 500 RPM for 1 minute. 10. Air dry for 20 minutes in the dark. 11. Resuspend in 7μl HiDi Formamide. 12. Spin plate briefly. 13. Seal with Costar foil. Store in freezer and plan to sequence within 3 days.

    • Can use tape instead of Costar foil. Just make sure to seal well.

Chelex Extraction Yeast Culture

1) Pellet 25.0ul of growing culture (1min at 15,000rpm) 2) Remove supernatent 3) Add 100.0ul of Chelex 4) Vortex and spin down 5) Boil for 10min 6) 1 min at 27C (to prevent lids from popping when opening) 7) Vortex and spin down at 15,000rpm for 1 min before use

Genus species NCYC # sample
Saccharomyces pastorianus 204 1
Saccharomyces kluyveri 543 2
Saccharomyces paradoxus 700 3
Saccharomyces dairenensis 777 4
Saccharomyces cerevisiae 1022 5
Saccharomyces servazzii 2307 6
Saccharomyces mikatae 2888 7
Saccharomyces cariocanus 2890 8
Saccharomyces spencerorum 2991 9
Saccharomyces humaticus 3083 10
Saccharomyces yakushimaensis 3084 11

MATA2 PCR of Chelex Extraction Yeast Culture

EX Takara RXN Mixture conc 1X 15
Milli Q water 6.15 92.25
Takara EX Buffer 10X 1 15
dNTP 2.5uM 0.8 12
Primer Mix 5uM 1 15
EX Taq 5U 0.05 0.75
Template Chelex Extraction 1
135
9.0ul / RXN and 1.0ul Chelex Extraction
Genus species NCYC # sample
Saccharomyces pastorianus 204 1
Saccharomyces kluyveri 543 2
Saccharomyces paradoxus 700 3
Saccharomyces dairenensis 777 4
Saccharomyces cerevisiae 1022 5
Saccharomyces servazzii 2307 6
Saccharomyces mikatae 2888 7
Saccharomyces cariocanus 2890 8
Saccharomyces spencerorum 2991 9
Saccharomyces humaticus 3083 10
Saccharomyces yakushimaensis 3084 11
Saccharomyces cerevisiae 1001 (+)
Negative Control (-)
PCR cycle
98C 10 sec
60C 1 min
x15

Load 2.0ul sample

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