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Team:Cambridge/Lab book/Week 12
From 2012.igem.org
| Week: | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 |
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Contents |
Monday (10/09/12)
- Attempted to run PCR of the backbone for the biobricks once more. Settings: Annealing temperature: 56°C, elongation time: 30secs.
- After running on gel, saw that while the -8 vector amplification appears to be producing some correctly sized (~2kb) bands, the other two only seem to be forming many primer dimers. Analysis of the sequence of the prefix and suffix revealed a CG palindrome that appears to be causing self annealing. Inserts are not affected, as they have the palindrome at the 5' end of the primer.
- Will retry at higher temperatures tomorrow.
- Attempted Gibson assembly for the -8 magnesium riboswitch biobrick with the backbone fragments produced earlier today and the insert fragments produced yesterday.
Transformation of e.coli with Gibson products
- E.coli cells transformed with Gibson products produced earlier today.
- Transformants plated out on 25μ/ml chloramphenicol plates.
Restriction digest of magnesium riboswitch construct plasmids
- Colonies grown up from colonies produced from Gibson products made on Check!!! were miniprepped and plasmid DNA extracted.
- Restriction digest performed with Sal1 and BamH1. Expected fragment sizes: 4900bp and 4500bp. If plasmid lacks insert: 4900bp and 3950bp. These two possibilities should be distinguishable.
- Correct banding pattern seen for all colonies grown up, with both -8 and +8 construct. Shall verify identity further with colony PCR.
Tuesday (11/09/12)
- Retried PCR of backbone for +8 magnesium riboswitch biobrick and fluoride biobrick at 60 °C and 64 °C.
- Verification gel showed that no bands of the appropriate size came out. Once more, only primer dimers produced.
- Production of primer dimers at these temperatures causes us to doubt that a successful PCR will be carried out at these temperatures. We may have to switch to ordinary ligation to construct our biobricks for submission.
Colony PCR of biobrick containing colonies
- Gibson assembly of biobrick plasmid yesterday produced a few colonies. These were tested with the standard sequencing primers to check if an insert of the correct size had been introduced into the bacteria.
- Gel showed that fragments of the appropriate size were produced for one of the -8 biobricks and one of the -8 colonies. These will be grown up, miniprepped and sent for sequencing.
Wednesday (12/09/12)
Thursday (13/09/12)
Magnesium Riboswitch Plate-Reader Assay
- Plate reader assay set up with magnesium riboswitch construct made over the last few days.
- Mg2+ concentrations used:
- 5μM
- 10μM
- 20μM
- 50μM
- 100μM
- 200μM
- 500μM
- 1mM
- 2mM
- 5mM
- 10mM
- 20mM
- IPTG concentrations used:
- 0.05mM
- 0.1mM
- 0.2mM
- 0.5mM
- 1mM
- 2mM
- 5mM
- 10mM
Friday (14/09/12)
Saturday (15/09/12)
Sunday (16/09/12)
