IGEM:Missouri University of Science and Technology/2009/Notebook/Cyanobacteria Mediated Filtration of Coal Flue/2013/08/06

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Date: 08/06/13 Blythe Ferriere, Annie Goo

Title: People in lab: Dilute primers plus PCR

Start Time: 2:15 PM

Purpose: Dilute primers for PCR reaction of norV gene, PCR norV gene from E. coli K-12 add iGEM prefix-suffix

Protocol: Dilution: Forward Primer: 0.39mg, To make 1 ug/uL: add 390 uL milliQ, To make 10ng/uL: add 1 uL of 1 ug/uL solution to 100 uL milliQ, Final Solution: ~0.7 uM Reverse Primer: 0.38mg, To make 1ug/uL: add 380 uL milliQ, To make 10ng/uL: add 1 uL of 1 ug/uL solution to 100 uL milliQ, Final Solution: ~0.7 uM

Products:

Sample Label Description Source Label Quantity
8/6/13 norV A 1 Amplified norV gene w/ iGEM prefix & suffix using 7.5 uL of template DNA template DNA 7/31/13 3
8/6/13 norV A 4 Amplified norV gene w/ iGEM prefix & suffix using 5 uL of template DNA Template DNA 7/31/13 3

Results: N/A

Notes: Changed programs about 15 minutes in

Stop Time: 5:00 PM

Next: Ligtate into iGEM backbone

Date: 08/06/13 Emily Puleo, Alie Abele

Title: Digestion of 8/3/13 GE norCB with EcoRI and PstI

Start Time: 6:17 PM

Purpose: Prep norCB for ligation with iGEM standard plasmid.

Protocol: LTM ed. 2 pg 37-39. Exceptions: 1. Master Mix: MilliQ - 39uL, EcoRI - 0.3uL, PstI - 0.3uL, 10x Tango buffer - 6uL, use 5uL DNA per reaction

Products:

Sample Label Description Source Label Quantity
8/6/13 D norCB digested with EcoRI and PstI 8/3/13 GE norCB 1
8/6/13 Master Master mix for digestion N/A 1

Results: N/A

Notes:

Stop Time: 9:00 PM

Next: Digestion of plasmids and ligation

Date: 08/06/13 Emily Puleo, Alie Abele

Title: PCR nosZ gene from P. aeruginosa to add iGEM prefix & suffix

Start Time: 7:32 PM

Purpose: To PCR amplify nosZ and add iGEM prefix and suffix

Protocol: LTM ed. 2 pg 48 Exceptions: 1. Final primer concentration <0.1 uM PCR run conditions: Annealing Temp: 65C, elongation time: 5min, template P. aeru 7/30/13

Products:

Sample Label Description Source Label Quantity
8/6/13 A nosZ nosZ gene with iGEM prefix&suffix using 7.5 uL template in 0.5 mL H2O P. aeruginosa 7/30/13 2
8/6/13 F primer nosZ 0.77 uM nosZ forward primer nosZ Forward Primer 1
8/6/13 R primer nosZ 0.8 uM nosZ reverse primer nosZ Reverse Primer 1

Results: N/A

Notes: See 7/31/13 notes for calculations

Stop Time: 9:00 PM

Next: Gel electrophoresis and sequencing

Date: 08/06/13 Emily Puleo, Alie Abele

Title: PCR hmp gene from E. coli to add iGEM prefix & suffix

Start Time: 8:10 PM

Purpose: To PCR amplify hmp and add iGEM prefix and suffix

Protocol: LTM ed. 2 pg 48 Exceptions: 1. Final primer concentration <0.1 uM PCR run conditions: Annealing Temp: 65C, elongation time: 5min, template washed and re-suspended E. coli 7/30/13

Products:

Sample Label Description Source Label Quantity
8/6/13 A hmp hmp gene with iGEM prefix&suffix using 7.5 uL template in 0.5 mL H2O E. coli 7/30/13 2
8/6/13 F primer hmp 10 ng/uL hmp forward primer hmp Forward Primer 1
8/6/13 R primer nosZ 10 ng/uL hmp reverse primer hmp Reverse Primer 1

Results: N/A

Notes: 0.39 mg of F primer + 390 uL of milliQ, diluted 1:100 with milliQ = 10 ng/uL F primer, repeated with 0.36 mg R primer and 360 uL milliQ. NOTE: Corrected math when entered into online notebook, recheck to confirm numbers

Stop Time: 9:00 PM

Next: Gel electrophoresis and sequencing


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