Team:Cambridge/LabBook/Week11
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Week 11: Monday 20th September - Sunday 26th September
Contents |
Monday
Tuesday
107. Expt: Biobrick assembly of pBAD+Luc (PP + LC) and (Emily)
- Plasmids already extracted
- Restriction digest following protocol on p88 using these quantities (in µl):
pBAD | PP Luc (1) | PP Luc (2) | LC Luc (1) | |
Nuclease-free H20 | 14 | 14 | 14 | 14 |
10x FD Buffer | 2 | 2 | 2 | 2 |
Plasmid DNA | 2 | 2 | 2 | 2 |
FD EcoRI | 1 | 1 | 1 | 1 |
FD SpeI | 1 | 0 | 0 | 0 |
FD XbaI | 0 | 1 | 1 | 1 |
- Gel electrophoresis
- pBAD failed - band at ~5000bp, should be 1200
- others look about right ~ between 3 & 5kb (should be 3600bp)
- Gel extraction - results in -20°C freezer
Wednesday
108. Expt: Send off PP + pSB1C3, EPIC pBAD for sequencing (Emily and Peter)
- Miniprep EPIC pBAD and nanodrop
- Prepare correct concentrations (100ng/µl for plasmid, 3.2pmol/µl for primers) to be sent off
109. Expt: Biobrick assembly of pBAD + PP Luc (1), PP Luc (2), LC Luc (1), PP+pSB1C3
- Peter 'miniprepped' PP+pSB1C3 to extract plasmid
- pBAD was amplified after trying to grow overnight culture failed. The following protocol was used:
Add to a PCR tube:
10µl | 2x Phusion Master Mix |
1µl | VF2 Primer |
1µl | VR Primer |
8µl | HPLC H20 |
20µl |
- Use stab to extract colony from pBAD plate and swirl in tube. Run following program on PCR machine saved as 'pBAD Amplification':
- Heated lid at 110°C
- Denaturation for 1m30s at 98°C
- Cycle 35 times
- Denaturation for 30s at 98°C
- Elongation for 2m at 72°C
- Elongation for 7m30s at 72°C
- PCR Purification of pBAD from PCR reaction was performed using Qiagen kit
- Restriction: repeating restriction of LC Luc (1) because tube from yesterday was dropped. Use protocol on p88 with following quantities (in µl):
pBAD | LC Luc (1) | |
HPLC H20 | 0 | 14 |
10x FD Buffer | 2 | 2 |
Plasmid DNA | 16 (PCR product)* | 2 |
FD EcoRI | 1 | 1 |
FD XbaI | 0 | 1 |
FD SpeI | 1 | 0 |
'*' Nanodrop said 29ng/µl after PCR Purification, which was strangely low. So used 16µl as recommended (in order to not exceed 0.2µg) but maximise amount of DNA.
- Gel electrophoresis: Run gel with following quantities:
pBAD | LC Luc (1) | |
DNA | 17 | 17 |
6x Orange LD | 3 | 3 |
µl°C