IGEM:Harvard/2008/Lab Notebooks/DailyBook/Week6/Chemical and Light: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
| Line 90: | Line 90: | ||
[[Image:7-29_gel_1_MXHTA.jpg]] | [[Image:7-29_gel_1_MXHTA.jpg]] | ||
=7/29 Mutagenesis of | =7/29 Mutagenesis of Cph1= | ||
We set up a reaction to remove the PstI site from cph1 (P87A and B) according to the QuikChange kit protocol. We ran a PCR positive control and a transformation positive control. We transformed all of the samples in XL 1-Blue cells and P87A in TOP10 as well. We used the TOP10 transformation protocol for both cell types. | We set up a reaction to remove the PstI site from cph1 (P87A and B) according to the QuikChange kit protocol. We ran a PCR positive control and a transformation positive control. We transformed all of the samples in XL 1-Blue cells and P87A in TOP10 as well. We used the TOP10 transformation protocol for both cell types. | ||
Revision as of 18:47, 29 July 2008
Sequencing Parts
Making Thermoinducible cI System
Minipreps from Transformations of P75 + P63 7/28
| Sample ID | ng/uL | A260 | 260/280 | 260/230 | Constant |
| E1 P3+mtrB notBB A | 60.36 | 1.207 | 1.95 | 1.99 | 50 |
| E1 P3+mtrB notBB B | 53.82 | 1.076 | 1.98 | 1.92 | 50 |
| E2 P1+mtrB A | 98.14 | 1.963 | 1.94 | 1.83 | 50 |
| E2 P1+mtrB B | 119.82 | 2.396 | 1.91 | 1.96 | 50 |
| E2 P3+mtrB NotBB A | 113.02 | 2.26 | 1.97 | 1.88 | 50 |
| E2 P3+mtrB NotBB B | 122.34 | 2.447 | 1.98 | 2.07 | 50 |
| PGW7 1A | 69.69 | 1.394 | 1.91 | 1.91 | 50 |
| PGW7 1B | 81.88 | 1.638 | 1.86 | 1.86 | 50 |
| PGW7 2A | 88.69 | 1.774 | 1.92 | 1.83 | 50 |
| PGW7 2B | 56.57 | 1.131 | 1.82 | 1.85 | 50 |
| P104A (S1 P75A+P63) | 66 | 1.32 | 1.74 | 0.81 | 50 |
| P104B (S1 P75B+P63) | 52.58 | 1.052 | 1.71 | 0.93 | 50 |
Digestion of Term + pLambda GFP System and cI857 7/28
| ' | P104a | cI857 RBS | cI857 BIG |
| DNA | 15 uL | 13 uL | 20 uL |
| 10x Buffer | 2.5 uL | 2.5 uL | 2.5 uL |
| 100x BSA | 0.25 uL | 0.25 uL | 0.25 uL |
| Restriction Enzyme 1 | 1 uL EcoRI | 1 uL EcoRI | 1 uL EcoRI |
| Restriction Enzyme 2 | 1 uL XbaI | 1 uL SpeI | 1 uL SpeI |
| Water | 5uL | 7uL | 0uL |
| Total | 25uL | 25uL | 25uL |
Western Blot to Test IPTG and Heat Induction of Lac Systems
Transformations in S1
Transformed mtrB constitutive system into S1 7/28
Electroporated and transformed S1.
| Plate | Marker | # Colonies | Description |
| E1 P3+mtrB notBB A | Kan | ||
| E1 P3+mtrB notBB B | Kan | ||
| E2 P1+mtrB notBB A | Kan | ||
| E2 P1+mtrB notBB B | Kan | ||
| E2 P3+mtrB NotBB A | Kan | ||
| E2 P3+mtrB NotBB B | Kan | ||
| (+) Ctrl (P59b) | Kan | ||
| (-) Ctrl (EB Buffer) | Kan |
RE digests 07/29
We digested several samples of P17 in the P1/P3 vector with EX. We will add the high/low constitutive promoters.
7/29 Mutagenesis of Cph1
We set up a reaction to remove the PstI site from cph1 (P87A and B) according to the QuikChange kit protocol. We ran a PCR positive control and a transformation positive control. We transformed all of the samples in XL 1-Blue cells and P87A in TOP10 as well. We used the TOP10 transformation protocol for both cell types.
Ligations of digested 7/26 minipreps
See last 2 gels of last week's notebook.
All cells are of E1 and were on Kan plates.
| DNA | Vector:Insert | # Colonies |
| P3A+(E1 P38+51) | 1:7 | 1 |
| P3A+(E1 P38+51) | 1:5 | 1 |
| P3A+(E1 P38+51) | 2:6 | 2 |
| P3A+(E1 P39+51 A) | 1:7 | 17 |
| P3A+(E1 P39+51 A) | 1:5 | 5 |
| P3A+(E1 P39+51 A) | 2:6 | 0 |
| P3A+(E1 P39+51 C) | 1:7 | 1 |
| P3A+(E1 P39+51 C) | 1:5 | 0 |
| P3A+(E1 P39+51 C) | 2:6 | 0 |
1-2 non-fluorescent colonies were picked for each sample with colonies.
