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

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dspB Track

Marianne and Vicki

Re-do PCR

  • No hot start; revised the amount of reagents used. Did not use PCR calculator
Table 1. Master Mix
REAGENT1 RXN VOLUME (uL)MASTER MIX (uL)
10x rxn buffer 2.5x922.5
10uM FW primer2x918
10uM RE primer2x918
10mM dNTP4x936
sdH2O9.3x983.7
Taq Polymerase0.2x91.8
DNA5x945
Total25x9225

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.


Table 2. PCR Tubes
1H49-His-A7H49-A
2H49-His-B8H49-B
3H80-His-A9H80-A
4H80-His-B10H80-B
5H171-His-A11H171-A
6H171-His-B12H171-B
13dH2O(control)
  • 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
  • Machine Conditions: 100V, 40mAmp, 45minutes, 1x TBE Buffer

Gel arrangement:

Table 3. Arrangement of gel
123456DNA 100bp ladder789101112


Results:
File:100623 DspB 2.tif

  • 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.
Table 4. Restriction Digest Supermix
REAGENTS1 RXN VOLUME (uL)SUPERMIX
Buffer 25x24120
BSA0.5x2412
ddH2O37x24888
Total42.5x241020
Table 5. Enzymes needed
ENZYMES1 RXN VOLUME (uL)TOTAL (uL)
XbaI1x2424
SpeI1x2424
Table 6. DNA Samples in Microcentrifuge Tubes
DNA SAMPLE1 RXN VOLUME (uL)
1RD4.7
2RD4.7
3RD4.7
4RD4.7
5RD4.7
6RD4.7
7RD4.7
8RD4.7
9RD4.7
10RD4.7
11RD4.7
12RD4.7
psB1C3 (backbone plasmid)4.7x12

Note: The number system is carried over from the labeling of the PCR tubes in the prior PCR reaction

  • Incubating at 37ºC. Left overnight.

Vicki Ma 03:31, 28 June 2010 (EDT)

Gel verification of above PCR products

See gel verification protocol in iGEM Training Manual Molecular Biology 2010. Written up on June 22 2010