IGEM:UBC/2009/Notebook/UBC iGEM 2010/2010/06/23

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Re-do PCR

 * No hot start; revised the amount of reagents used. Did not use PCR calculator

Protocol: Two Master Mixes:
 * 1) Master Mix C (MMC): FW primer is His6-tag primer
 * 2) Master Mix D (MMD): FW primer does NOT have His6-tag

Step 1: Pipet all of the above (in table 1) [20uL] except for gDNA template into appropriately labeled PCR tubes.
 * Another change: doing doubles instead of triples

Step 2: PCR Cycles
 * 95ºC @ 2 min (Denaturing)
 * Cycle Program 30x:
 * 95ºC @ 30s (Denaturing)
 * 65ºC @ 10s (Annealing)
 * 72ºC @ 80s (Extension)  *elongation time changed 
 * 72ºC @ 10 min (Final extension)
 * 10ºC @ Hold (Chill)

Start time for PCR: 1023 End time for PCR: 1151

Gel Verification of above PCR Products
See gel verification protocol in iGEM Training Manual Molecular Biology 2010. Protocol written up here: June 22 2010
 * Changes to protocol:
 * Used 10uL of DNA (no dilution with dH2O) with 2uL of loading dye for a total of 12uL

Gel arrangement: Results:
 * Machine Conditions: 100V, 40mAmp, 45minutes, 1x TBE Buffer
 * Distinct bands can be seen for H171, at ~1500bp and above 1500bp
 * No distinct bands can be seen for H49, or H80

Restriction Digestion
Protocol: Refer to "iGEM Common Protocols" Supplies Needed: Restriction digest supermix (5*n μl of Buffer 2, 0.5*n μl of BSA, 37*n μl of ddH2O where n = # of 50uL reactions) stored at -20°C Enzymes DNA

Steps:
 * 1) Thaw restriction digest supermix (42.5μl aliquots).
 * 2) Add 0.3-0.5μg DNA (1-5uL)
 * 3) Add 1uL of each restriction enzyme.
 * 4) Incubate at 37°C for 2 hours.

Note: The number system is carried over from the labeling of the PCR tubes in the prior PCR reaction Vicki Ma 03:31, 28 June 2010 (EDT)
 * Incubating at 37ºC. Left overnight.

Gel verification of above PCR products
See gel verification protocol in iGEM Training Manual Molecular Biology 2010. Written up on June 22 2010