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Team:Hawaii/Notebook/2008-09-18
From 2008.igem.org
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:* PCR of 3A:nrsg and 3A:nrpg13-8 plasmids | :* PCR of 3A:nrsg and 3A:nrpg13-8 plasmids | ||
| + | |||
| + | ===Construction of Secretion Device=== | ||
| + | :<strong>Krystle</strong> | ||
| + | |||
| + | :3A ligation of cut pSB1A3 with the following inserts: | ||
| + | ::* nir + gfpf + tt #8 | ||
| + | ::* nir + gfpf + tt #17 | ||
| + | ::* nir + gfpf + tt #21 | ||
| + | ::* slr + gfpf + tt #8 | ||
| + | ::* slr + gfpf + tt #17 | ||
| + | ::* slr + gfpf + tt #21 | ||
===Restreaked BB-pRL1383a plate=== | ===Restreaked BB-pRL1383a plate=== | ||
Latest revision as of 01:40, 21 September 2008
| Projects | Events | Resources | ||
|---|---|---|---|---|
| Sponsors | Experiments | Milestones | Protocols | |
| Notebook (t) | Meetings (t) |
Things we did today
Wetlab work
Plasmid prep
- Grace
- Inoculated TB+sp100 with BB-pRL1383a for plasmid prep
PCR to verify plasmid preps
- Grace
- PCR of 3A:nrsg and 3A:nrpg13-8 plasmids
Construction of Secretion Device
- Krystle
- 3A ligation of cut pSB1A3 with the following inserts:
- nir + gfpf + tt #8
- nir + gfpf + tt #17
- nir + gfpf + tt #21
- slr + gfpf + tt #8
- slr + gfpf + tt #17
- slr + gfpf + tt #21
Restreaked BB-pRL1383a plate
- Grace
Transformation
Margaret
- transformed ligation products in DH5-alpha
Drylab Work
Sequencing Analysis
- Grace
- Correct:
- C0012V (sequence to see what it was): EcoRI/XbaI/SpeI/PstI sites present. Insert aligns to sequence for lacI (C0012 insert, vector was uncut)
- C0012V-SAP'd (sequence to see what it was): EcoRI/XbaI/SpeI/PstI sites present. Insert aligns to sequence for lacI (C0012 insert, vector was uncut)
- slr1+GFPf
- Incorrect:
- 3A:nrp: EcoRI/PstI sites present. 82bp insert aligns to sequence for E. coli flagellar biosynthesis RNA polymerase sigma factor
- 3A:nrpg12-2: EcoRI/PstI sites present. 759bp insert aligns to sequence for E. coli fumarate reductase (anaerobic) catalytic and NAD/flavoprotein subunit
- 3A:nrpg12-6: EcoRI/PstI sites present. 899bp insert aligns to sequence for E. coli protein C5 component of RNAse P
- 3A:nrs2: EcoRI/PstI sites present. 85bp insert aligns to sequence for E. coli diaminopimelate decarboxylase, PLP-binding
- 3A:nrs3: SpeI/PstI sites present. Insert aligns to pilA sequence and pSB1A3 transcriptional termination stem loop
- 3A:nrsg10: SpeI/PstI sites present. Insert aligns to pilA + GFPf
- pilA+GFPf12: GFPf only
- plac+rbs1 (sequence to see what it was): EcoRI/XbaI/SpeI/PstI sites present. rbs (B0034) only
- plac+rbs2 (sequence to see what it was): EcoRI/XbaI/SpeI sites present. Insert showed 97% similarity to RP4 origin of replication
- plac+rbs12 (sequence to see what it was): EcoRI/XbaI/SpeI/PstI sites present. Insert is GFP only.
- Resequence:
- plac+rbs (no read -- only 5bp sequenced ?!?)
- pilA+GFPf13 (no read -- only 5bp sequenced ?!?)
Discussion
- Please throw away old plates in 4C fridge. Some have fungi growing on them already.
Quote of the Day
History is the only laboratory we have in which to test the consequences of thought. - Étienne Gilson
[http://manoa.hawaii.edu/ 
][http://manoa.hawaii.edu/ovcrge/ 
][http://www.ctahr.hawaii.edu 
]
