IGEM:Harvard/2008/Lab Notebooks/DailyBook/Week6/Chemical and Light: Difference between revisions
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===Gel of Digestion 7/29=== | |||
Gel here-- P104 was not cut. | |||
==Digestion of P104a with new XbaI 7/29== | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''P104a''' | |||
|- | |||
| DNA ||20 uL | |||
|- | |||
| 100x BSA||0.25 uL | |||
|- | |||
| 10x Buffer||2.5 uL | |||
|- | |||
| EcoRI||0.5 uL | |||
|- | |||
| XbaI||1 uL | |||
|- | |||
| Water||0.75 uL | |||
|- | |||
| Volume||25 uL | |||
|} | |} | ||
Revision as of 19:46, 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 |
Gel of Digestion 7/29
Gel here-- P104 was not cut.
Digestion of P104a with new XbaI 7/29
' | P104a |
DNA | 20 uL |
100x BSA | 0.25 uL |
10x Buffer | 2.5 uL |
EcoRI | 0.5 uL |
XbaI | 1 uL |
Water | 0.75 uL |
Volume | 25 uL |
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 |
Housekeeping
Made S1 Electrocompetent Cells 7/29
Made ~250 aliquots of wildtype and mtrB- Shewie-- 80 uL in each aliquot.
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.
Lane 1: 1 kb ladder
Lane 2: uncut E2 P3+17 B (3652)
Lane 3: E1 P1+17 A cut EX (3652)
Lane 4: E1 P1+17 B cut EX (3652)
Lane 5: E1 P1+17 C cut EX (3652)
Lane 6: E1 P1+17 D cut EX (3652)
Lane 7: E2 P1+17 A cut EX (3652)
Lane 8: E2 P1+17 B cut EX (3652)
Lane 9: E2 P3+17 A cut EX (3652)
Lane 10: E2 P3+17 B cut EX (3652)
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.