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Team:Hawaii/Notebook/2008-09-12
From 2008.igem.org
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===Verification of transformants=== | ===Verification of transformants=== | ||
:<strong> Grace</strong> | :<strong> Grace</strong> | ||
| - | [[Image:091208colonyPCR. | + | [[Image:091208colonyPCR.png|right|thumb|400px|EtBr stained 2% agarose gel ran at 95V for 1 hour. Two microliters of PCR product were loaded into each well.]] |
{|class=wikitable border=1 align=center | {|class=wikitable border=1 align=center | ||
!Construct | !Construct | ||
| Line 64: | Line 64: | ||
|align=center|301 | |align=center|301 | ||
|} | |} | ||
| + | :* Colony PCR of 3A and plac+rbs back ligations colonies | ||
| + | ::* Restreaked: | ||
| + | :::* 3A:nir+rbs+slr1 colonies 2 & 3 | ||
| + | :::* 3A:nir+rbs+pilA colony 6 | ||
| + | :::* 3A:nir+rbs+slr1+GFPf colony 10 | ||
| + | :::* 3A:nir+rbs+pilA+GFPf12 colonies 2 & 6 | ||
| + | :::* 3A:nir+rbs+pilA+GFPf13 colonies 4, 6, 8, 10 | ||
===Competent Cells=== | ===Competent Cells=== | ||
| Line 92: | Line 99: | ||
===ON Culture of B_H_R plasmid parts=== | ===ON Culture of B_H_R plasmid parts=== | ||
| - | <strong>Margaret</strong> | + | :<strong>Margaret</strong> |
:*5ml ON culture, LB with Amp100 for: colonies with the correct insert from a transformation completed on 9/7. | :*5ml ON culture, LB with Amp100 for: colonies with the correct insert from a transformation completed on 9/7. | ||
| - | {|class=wikitable border=1 | + | {|class=wikitable border=1 align=center |
|Construct | |Construct | ||
|# colonies checked | |# colonies checked | ||
| Line 113: | Line 120: | ||
|2, 3, 4, 5, 6, 8 | |2, 3, 4, 5, 6, 8 | ||
|} | |} | ||
| - | |||
| - | |||
| - | |||
= Discussion = | = Discussion = | ||
Latest revision as of 08:44, 15 September 2008
| Projects | Events | Resources | ||
|---|---|---|---|---|
| Sponsors | Experiments | Milestones | Protocols | |
| Notebook (t) | Meetings (t) |
Things we did today
Wetlab work
Verification of transformants
- Grace
| Construct | Colony forming units |
|---|---|
| 3A: nir+rbs + slr1 | 4 |
| 3A: nir+ rbs + pilA | 32 |
| 3A: nir+rbs + slr1+GFPf | 35 |
| 3A: nir+rbs + pilA+GFPf (8/12) | 42 |
| 3A: nir+rbs + pilA+GFPf (8/13) | 24 |
| pSB1A3 (-) | 25 |
| plac+rbs + slr1 | >300 |
| plac+rbs + pilA | >300 |
| plac+rbs + slr1+GFPf | >300 |
| plac+rbs + pilA+GFPf (8/12) | >300 |
| plac+rbs + pilA+GFPf (8/13) | >300 |
| plac+rbs (-) | 295 |
| nir+rbs + slr1 | >300 |
| nir+rbs + pilA | >300 |
| nir+rbs + slr1+GFPf | >300 |
| nir+rbs + pilA+GFPf (8/12) | >300 |
| nir+rbs + pilA+GFPf (8/13) | >300 |
| nir+rbs (-) | 301 |
- Colony PCR of 3A and plac+rbs back ligations colonies
- Restreaked:
- 3A:nir+rbs+slr1 colonies 2 & 3
- 3A:nir+rbs+pilA colony 6
- 3A:nir+rbs+slr1+GFPf colony 10
- 3A:nir+rbs+pilA+GFPf12 colonies 2 & 6
- 3A:nir+rbs+pilA+GFPf13 colonies 4, 6, 8, 10
Competent Cells
- Margaret, Krystle, & Grace
- Several attempts were made to make more DH5-alpha competent cells.
- 5ml culture grown over-night in TB (terrific broth), in the morning 125ml SOB was inoculated with 12.5mL(1), and 2 flasks with 50ml (2&3) SOB were inoculated with 5ml of the ON culture.
| Flask # | OD550 (1hr) | OD550 (1.5hr) |
|---|---|---|
| 1 | 0.2056 | 0.3432 |
| 2 | 0.2006 | 0.3312 |
| 3 | 0.2547 | 0.4184 |
- The cells were spun down at 2147 x g, 4°C, 30 minutes. The yield was low (1/4 of the tip of a 50ml centrifuge tube), so the culturing was repeated, except that the starting OD was 0.14 and the final OD was more than 0.3 but less than 0.6 (with the same amount of cells yielded).
- The cells are being cultured again tonight to try again tomorrow. IF we get the same amount of cells, we will continue with the competent cell making procedure.
ON Culture of B_H_R plasmid parts
- Margaret
- 5ml ON culture, LB with Amp100 for: colonies with the correct insert from a transformation completed on 9/7.
| Construct | # colonies checked | correct (these are plasmid prepped) |
| pSB1A3::plac+RBS(B0030)+rep | 8 | 0 |
| pSB1A3::plac+RBSB0034+aadA(prl1383a)colony 1 | 8 | 2, 6 |
| pSB1A3::plac+RBSB0034+aadA(BB)colony9 | 8 | 2, 3, 4, 5, 6, 8 |
Discussion
Quote of the Day
Wait, you have an internet boyfriend? - KS, to Norman
[http://manoa.hawaii.edu/ 
][http://manoa.hawaii.edu/ovcrge/ 
][http://www.ctahr.hawaii.edu 
]
