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
| REAGENT | 1 RXN VOLUME (uL) | | MASTER MIX (uL) |
| 10x rxn buffer | 2.5 | x9 | 22.5 |
| 10uM FW primer | 2 | x9 | 18 |
| 10uM RE primer | 2 | x9 | 18 |
| 10mM dNTP | 4 | x9 | 36 |
| sdH2O | 9.3 | x9 | 83.7 |
| Taq Polymerase | 0.2 | x9 | 1.8 |
| DNA | 5 | x9 | 45 |
| Total | 25 | x9 | 225 |
Protocol:
Two Master Mixes:
- Master Mix C (MMC): FW primer is His6-tag primer
- 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
| 1 | H49-His-A | 7 | H49-A |
| 2 | H49-His-B | 8 | H49-B |
| 3 | H80-His-A | 9 | H80-A |
| 4 | H80-His-B | 10 | H80-B |
| 5 | H171-His-A | 11 | H171-A |
| 6 | H171-His-B | 12 | H171-B |
| 13 | dH2O(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
| 1 | 2 | 3 | 4 | 5 | 6 | DNA 100bp ladder | 7 | 8 | 9 | 10 | 11 | 12 |
Results:
- 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:
- Thaw restriction digest supermix (42.5μl aliquots).
- Add 0.3-0.5μg DNA (1-5uL)
- Add 1uL of each restriction enzyme.
- Incubate at 37°C for 2 hours.
Table 4. Restriction Digest Supermix
| REAGENTS | 1 RXN VOLUME (uL) | SUPERMIX |
| Buffer 2 | 5 | x24 | 120 |
| BSA | 0.5 | x24 | 12 |
| ddH2O | 37 | x24 | 888 |
| Total | 42.5 | x24 | 1020 |
Table 5. Enzymes needed
| ENZYMES | 1 RXN VOLUME (uL) | TOTAL (uL) |
| XbaI | 1 | x24 | 24 |
| SpeI | 1 | x24 | 24 |
Table 6. DNA Samples in Microcentrifuge Tubes
| DNA SAMPLE | 1 RXN VOLUME (uL) |
| 1RD | 4.7 |
| 2RD | 4.7 |
| 3RD | 4.7 |
| 4RD | 4.7 |
| 5RD | 4.7 |
| 6RD | 4.7 |
| 7RD | 4.7 |
| 8RD | 4.7 |
| 9RD | 4.7 |
| 10RD | 4.7 |
| 11RD | 4.7 |
| 12RD | 4.7 |
| psB1C3 (backbone plasmid) | 4.7 | x12 | 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
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