IGEM:Harvard/2008/Lab Notebooks/DailyBook/Week10/Chemical and Light: Difference between revisions
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=Getting Thermoinducible and Lac Inducible GFP into pSC101= | =Getting Thermoinducible and Lac Inducible GFP into pSC101= | ||
==Digests of Inducible Systems and pSC101 8/27== | |||
P102 needs to be retransformed so it was not included in this set. | |||
===Gel of Digest 8/27=== | |||
{| {{table}} | |||
!rowspan=11|[[Image:8-27-08_amy_digests.jpg]] | |||
!colspan=2 align="center" style="background:#f0f0f0;"|'''1% agarose''' | |||
|- | |||
| align="center" style="background:#f0f0f0;"|'''Lane''' | |||
| align="center" style="background:#f0f0f0;"|'''Sample''' | |||
|- | |||
| 1||1 kb + DNA Ladder | |||
|- | |||
| 2||P124 1:2.5 A | |||
|- | |||
| 3||P124 1:2.5 B | |||
|- | |||
| 4||P125 1:2.5 Dephos A | |||
|- | |||
| 5||P125 1:2.5 D | |||
|- | |||
| 6||mtrB TOPO 0.5 uL B | |||
|- | |||
| 7||mtrB TOPO 2 uL B | |||
|- | |||
| 8||mtrB TOPO 4-1 | |||
|- | |||
| 9||mtrB TOPO 4-10 | |||
|} | |||
mtrB TOPO bands were cut (2.2 kB) but P124-5 did not cut properly, suggesting that there was an extra cut site that was cutting the insert into two pieces. | |||
===Single-cut Digest of P124-5 8/27=== | |||
=Getting mtrB onto thermo and lac inducible systems= | =Getting mtrB onto thermo and lac inducible systems= | ||
Revision as of 22:38, 30 August 2008
Transformations in Shewie
About 250 ng of DNA added each time.
First Set 8/22
| Plate | Colonies | Size | Color |
| S1 P102 (P108+45) Kan | 5 colonies | 3mm to 5 mm | Pink |
| S1 P121+P38 (P124) 1:2.5 Kan A | 1 colony | 5mm to 7.5mm | Pink |
| S1 P28 (ColE1) Kan | 0 colonies | ||
| S1 P121+P38(P124) 1:2.5 Kan B | 0 colonies | ||
| S1 P121+P39 1:2.5 Dephos Kan A | 0 colonies | ||
| S1 Topo vector (puc) 2ul Kan X-gal | >50 colonies | 1mm | White |
| S1 Topo vector (puc) 4ul Kan X-gal | >50 colonies | 1mm | White |
| S1 P121+P39 1:2.5 Kan D | 0 colonies | ||
| S1 (-) ctrl (just cells) Kan | 0 colonies | ||
| S1 (+) ctrl (E1 p59b) Kan | 2 colonies | 5mm | Pink |
Poor transformation efficiency in the positive control suggests that there may be a problem with our cells. All samples, except for P102, were retransformed the following week with fresh cell cultures.
Second Set (with new cells) 8/27
| Plate | Marker | Description |
| S1 P124 1:2.5 A | Kan | 14 2-3mm pink colonies, ~100 0.5mm white colonies |
| S1 P124 1:2.5 B | Kan | 2 pink 2mm colonies, >100 0.5mm white colonies |
| S1 P125 1:2.5 Dephos A | Kan | 2 pink 2mm colonies, >100 0.5mm white colonies |
| S1 P125 1:2.5 D | Kan | 8 2mm pink colonies, ~100 0.5mm white colonies |
| S1 P28 | Kan | ~8 1-2mm pink colonies, ~50 0.5mm white colonies |
| S1 TOPO 2uL | Kan | Lawn of (>200) 0.5mm white colonies |
| S1 TOPO 4 uL (w/ Xgal) | Kan | Lawn of (>200) 0.5mm white colonies |
| S1 P59b (+) ctrl | Kan | ~50 2mm pink colonies, ~50 0.5mm white colonies |
| S1 (-) ctrl | Kan | 50-100 0.5mm white colonies |
Getting Thermoinducible and Lac Inducible GFP into pSC101
Digests of Inducible Systems and pSC101 8/27
P102 needs to be retransformed so it was not included in this set.
Gel of Digest 8/27
|
1% agarose | |
|---|---|---|
| Lane | Sample | |
| 1 | 1 kb + DNA Ladder | |
| 2 | P124 1:2.5 A | |
| 3 | P124 1:2.5 B | |
| 4 | P125 1:2.5 Dephos A | |
| 5 | P125 1:2.5 D | |
| 6 | mtrB TOPO 0.5 uL B | |
| 7 | mtrB TOPO 2 uL B | |
| 8 | mtrB TOPO 4-1 | |
| 9 | mtrB TOPO 4-10 | |
mtrB TOPO bands were cut (2.2 kB) but P124-5 did not cut properly, suggesting that there was an extra cut site that was cutting the insert into two pieces.
Single-cut Digest of P124-5 8/27
Getting mtrB onto thermo and lac inducible systems
Ligating mtrB TOPO with RBS
Grew up more mtrB TOPO for next week.
PCR of mtrB with RBS + mtrB primers
Scheduled for next week.
