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Team:Groningen/Notebook/28 August 2009
From 2009.igem.org
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Wet
GVP Cluster
- → DONE Isolate plasmids from o.n. cultures
- → TODO Restriction analysis of plasmids for correct ligation of GVP behind LAC/pBAD promoter
- → TODO Test control of bouyancy in Saline solution
- → TODO Order synthetic DNA for GVP
- → TODO Test promoter strenght compared to BBa_J23101 promoter (Sven)
- → TODO Enter sequences of constructs to Sandbox
Overnight Precultures
All tubes showed growth of E.coli cells, and must have ampicillin resistance on a plasmid(The tubes differed in cell density though). To be sure of correct ligation products in the plasmids an isolation will be performed, followed by restriction analysis. If positive, the plasmids will be sent for sequencing.
Overnight Plates
The plates grown overnight for glycerol stocks showed single colony growth on the second and third wipe stripes. The plates were stored in the fridge for preculture inoculation on monday (31/8).
- → The colonies for pArsR+RBS showed red colonies, and the pCueO+RBS/pZntR+RBS showed normal yellow/white colonies.
Restriction for Assembly
The vector pSB1AC3 containing the LAC/pBAD inducible promoters and GVP were cut with PstI and EcoRI to check correct ligation product.
- 5μL plasmid in MQ
- 11μL MQ (end volume of 20μL)
- 2μL Fast digest buffer
- 1μL PstI fast digest enzyme
- 1μL EcoRI fast digest enzyme
Restriction was kept at 37C for 30 min., followed by addition of 6x loading dye and put on ice until used for gel analysis.
- → pLacI
- → pSB1A2
Observations
- → Tests for bouancy were thought to have been carried out in Saline solution, but instead it was a 1000 times diluted solution. The recipe for Saline is as follows:
- 9 grams NaCl in 1 liter H2O (1x solution, 0.9% NaCl w/v)
- 90 grams NaCl in 1 liter H2O (10x soltion)
- → The pBad/araC and pLacI were thought to be transfered to the pSB1AC3 plasmids, and out of the pSB1A2 plasmid. Restriction analysis (used for assembly) showed fragments of the size of pSB1A2, and not pSB1AC3 plasmid. The sequencing results should give desisive prove if it is a correct transfer or re-(self)ligation back into its original plasmid pSB1A2.
Transporters
HmtA
The overnight restriction and the pcr show that only sample 7 can be a correct construct. NcoI will cut 2 times resulting in 2 fragments. One of 1814bp and one of 3261bp. The pcr should result in a grafmetn of 2336bp. this gel does show a convincing cut. but not the expected pcr product.
Metal Accumulation
Vectors
Dry
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