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Team:Cambridge/Week 4/Lab book
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Contents |
Monday
Transformation of bacillus and e.coli with lux genes from 2010
Tuesday
Induction of lux genes in e.coli with IPTG
- Plasmid PJS 130 used as our backbone for isolating the magnesium riboswitch.
- Primers:
- Forward: TTCAAAACATGACCTATGACgtcgcagagtatgccg
- Reverse: cctccctctgctaaaacACAAGGCATTAACACTACAT
- PCR settings:
- 95 °C - 6mins
- 98 °C - 10secs
- 58 °C - 45secs
- 72 °C - 180secs
- Repeat above 35x
- 72 °C - 5mins
- 25 °C - 1min
PCR of riboswitch DNA from bacillus genome
- Colony of strain check what the strain was used as template for this experiment.
- Primers used:
- Forward: cggagggagacgattttgTGTTCCGTAATTGTGATGTAAG
- Reverse: acaccggcatactctgcgacGTCATAGGTCATGTTTTGAACC
- PCR settings - as above (run in parallel).
Wednesday
Gel electrophoresis of PCR products
- PCR fragments for Mg2+ riboswitch from yesterday separated on gel. 90mins at 100V.
- Lane 1: Ladder
- Lane 2 + 3: Riboswitch DNA from genome (replicates) - 550bp fragment expected.
- Lane 4 + 5: Vector DNA from pJS130 (replicates)- Chloramphenicol resistance marker - 9.0kbp fragment expected
- Lane 6: Postitive control - 1.9kbp fragment expected
- Riboswitch DNA was successfully amplified. However, vector DNA was not. Given that the postive control was also successful, it seems likely that something went wrong with some stage before the PCR amplification. We will try to rectify the problem tomorrow.
Purification of riboswitch DNA from gel
- Successful riboswitch DNA extracted from gel and purified using a minelute column. DNA frozen for later Gibson assembly.
Thursday
- Plasmid PJS 130 used as our backbone for isolating the magnesium riboswitch.
- Primers:
- Forward: TTCAAAACATGACCTATGACgtcgcagagtatgccg
- Reverse: cctccctctgctaaaacACAAGGCATTAACACTACAT
- PCR settings:
- 95 °C - 6mins
- 98 °C - 10secs
- ~55 °C - 45secs
- 72 °C - 240secs
- Repeat above 35x
- 72 °C - 5mins
- 25 °C - 1min
- Settings changed - Annealing temperature and elongation time - attempting to debug PCR.
Gel electrophoresis of PCR products
- PCR fragments for Mg2+ riboswitch from yesterday separated on gel. 90mins at 100V.
- Lane 1: Ladder
- Lane 2: Vector DNA from pJS130 @ °C - 9.0kbp fragment expected
- Lane 3: Vector DNA from pJS130 @ °C - 9.0kbp fragment expected
- Lane 4: Vector DNA from pJS130 @ °C - 9.0kbp fragment expected
- Lane 5: Postitive control - 1.9kbp fragment expected
- Lane 6: Negative control - no fragments expected
- Riboswitch DNA was successfully amplified. However, vector DNA was not. Given that the postive control was also successful, it seems likely that something went wrong with some stage before the PCR amplification. We will try to rectify the problem tomorrow.
