IGEM:Harvard/2008/Lab Notebooks/DailyBook/Week10/Chemical and Light: Difference between revisions
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Further inspection of the sequence revealed that there was an extra PstI site after the cI repressor coding region due to an error that was made while making the primers that resulted in the suffix being backward and the cut sites on the part to read EXPS. Attempts to mutate out the extra site and basepairs will begin next week once primers are reviewed and ordered-- might take a while just to get them. | Further inspection of the sequence revealed that there was an extra PstI site after the cI repressor coding region due to an error that was made while making the primers that resulted in the suffix being backward and the cut sites on the part to read EXPS. Attempts to mutate out the extra site and basepairs will begin next week once primers are reviewed and ordered-- might take a while just to get them. | ||
Due to the extra PstI site, all future ligations involving the thermoinducible system cut with PstI will need to be held off (ie. getting into the pSC101 vector, and adding on mtrB or mtrA) | Due to the extra PstI site, all future ligations involving the thermoinducible system cut with PstI will need to be held off (ie. getting into the pSC101 vector, and adding on mtrB or mtrA) until the issue is resolved either with site directed mutagenesis or by redesigning primers for cI and starting over? | ||
=Getting mtrB onto thermo and lac inducible systems= | =Getting mtrB onto thermo and lac inducible systems= | ||
Revision as of 22:47, 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
|
1.2% E-gel visualized using EtBr/UV | |
|---|---|---|
| Lane | Sample | |
| 1 | 1 kb + DNA Ladder | |
| 2 | P124 1:2.5 A with XbaI | |
| 3 | P124 1:2.5 B with XbaI | |
| 4 | P125 1:2.5 Dephos A with XbaI | |
| 5 | P125 1:2.5 D with XbaI | |
| 7 | P124 1:2.5 A with PstI | |
| 8 | P124 1:2.5 B with PstI | |
| 9 | P125 1:2.5 Dephos A with PstI | |
| 10 | P125 1:2.5 D with PstI | |
| 11 | 1 kb + DNA Ladder | |
IMPORTANT NOTE Further inspection of the sequence revealed that there was an extra PstI site after the cI repressor coding region due to an error that was made while making the primers that resulted in the suffix being backward and the cut sites on the part to read EXPS. Attempts to mutate out the extra site and basepairs will begin next week once primers are reviewed and ordered-- might take a while just to get them.
Due to the extra PstI site, all future ligations involving the thermoinducible system cut with PstI will need to be held off (ie. getting into the pSC101 vector, and adding on mtrB or mtrA) until the issue is resolved either with site directed mutagenesis or by redesigning primers for cI and starting over?
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.
