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Reporter: Week 5 June 12-17
From 2011.igem.org
Contents |
Sunday
Mutagenesis of XylE, Take 3 Day 4
The PCR product of the mutated XylE gene from the 4°C fridge was purifed, transformed into Escherichia coli cells and plated onto ampicillin resistant plates.
Monday
Insert tev Protease into K3 Vector, Day 4
Sequencing Results: The tev protease sequence came back 99% correct. The assembly features a single point mutation at base pair 515: TCT (serine) -> CCT (proline).
Insert Imperial Linker into K3 Vector, Day 4
Since we failed to make freezer stock of this correctly assembled part, we transformed the Imp linker + K3 miniprep made for sequencing on Friday, 6/10 into Escherichia coli cells and plated onto kanamycin resistant plates.
Mutagenesis of XylE, Take 4 Day 1
The modified Knight Lab Multi site PCR protocol outlined on Friday, 6/10 was followed for the mutagenesis of the XylE gene. No product was seen on the gel, meaning that the mutagenesis did not work. An alternate PCR for mutagenesis protocol, which contained the following changes from the protocol outlined on Friday, 6/10, was performed.
| Material Added | Listed Volume Added on 6/10(μl) | Specifically Modified Volume Added (μl) |
|---|---|---|
| primer 486 | 0.3 | 0.5 |
| primer 837 | 0.3 | 0.5 |
| dH2O | 17.5 | 18 |
NOTE: This PCR reaction only includes two primers, listed above, because these primers have similar annealing temperatures (79.54°C and 79.38°C) while the third primer has a higher annealing temperature (84.32°C).
Insert cI and tev Cleavage Sites into K3 Vector, Day 1
Since the Imp linker part in the K3 vector contains all of the restriction sites (EcoRI, XbaI, PstI, SpeI, AgeI) the cI and tev cleavage sites were inserted into the K3 vector using the Imp linker + K3 part. The Imp linker + K3 miniprep created Friday, 6/10 and the purified cI and tev cleavage site PCR products from 6/3 were digested with the XbaI and AgeI restriction enzymes in buffer 4. The cI cleavage site and the tev cleavage site were ligated with the Imp linker + K3 construct. These ligations were transformed into Escherichia coli cells and plated onto kanamycin resistant plates.
