IGEM:Harvard/2008/Lab Notebooks/DailyBook/Week6/Chemical and Light: Difference between revisions
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|- | |- | ||
| Total||21 uL||21 uL||21 uL||21 uL||21 uL||21 uL | | Total||21 uL||21 uL||21 uL||21 uL||21 uL||21 uL | ||
|} | |||
Transformed in E1. | |||
===Plates from Transformations 8/2=== | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Plate''' | |||
| align="center" style="background:#f0f0f0;"|'''Marker''' | |||
| align="center" style="background:#f0f0f0;"|'''# Colonies''' | |||
| align="center" style="background:#f0f0f0;"|'''Description''' | |||
|- | |||
| E1 P75a + P63b||Kan|||| | |||
|- | |||
| E1 P75b + P63b||Kan|||| | |||
|- | |||
| E1 P75a + P63b||Kan|||| | |||
|- | |||
| E1 P75b + P63b||Kan|||| | |||
|- | |||
| E1 P75a + P63b||Kan|||| | |||
|- | |||
| E1 P75b + P63b||Kan|||| | |||
|- | |||
| puc19 + ctrl||Amp|||| | |||
|- | |||
| (-) ctrl||Kan|||| | |||
|} | |} | ||
Revision as of 22:05, 1 August 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 cut correctly but I made a mistake and thought it hadn't been cut properly, so discarded gel and repeated digest.
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 |
Gel for Digestion 7/30
|
1% Agarose, visualized using EtBr/UV | |
|---|---|---|
| Lane | Sample | |
| 1 | 1 kB Ladder | |
| 2 | P104a (3675bp) | |
| 3 | 100bp Ladder | |
P104 was extracted and gel purified. It was dephosphorylated with the rAPid Alkaline Phosphatase.
Ligation of P104 and cI 7/30
| ' | P104a + RBS | P104a + BIG | P104a + RBS | P104a + BIG |
| Insert | 6 | 6 | 5 | 5 |
| Vector | 2 | 2 | 1 | 1 |
| Dilution Buffer | 2 | 2 | 2 | 2 |
| Water | 0 | 0 | 2 | 2 |
| Total | 10 | 10 | 10 | 10 |
| Then add: | ||||
| Ligation Buffer | 10 | 10 | 10 | 10 |
| Ligase | 1 | 1 | 1 | 1 |
| Total | 21 uL | 21 uL | 21 uL | 21 uL |
Transformed E1 with 5 uL of each ligation reaction.
Plate Results 7/31
| Plate | Marker | # Colonies | Description |
| P104a + RBS (6:2) | Kan | 0 | |
| P104a + BIG (6:2) | Kan | 0 | |
| P104a+ RBS (5:1) | Kan | 0 | |
| P104a + BIG (5:1) | Kan | 0 | |
| (+) Ctrl (puc19) | Amp | 25-50 | Not very evenly distributed. |
| (-) Ctrl (EB buffer) | Kan | 0 |
Re-Ligation of P104a + cI 7/31
Used the same purified fragments as before and ligated again in TOP10 cells.
| ' | P104a + RBS | P104a + BIG |
| Insert | 6 | 6 |
| Vector | 2 | 2 |
| Dilution Buffer | 2 | 2 |
| Water | 0 | 0 |
| Total | 10 | 10 |
| Then add: | ||
| Ligation Buffer | 10 | 10 |
| Ligase | 1 | 1 |
| Total | 21 | 21 |
Plate Results 8/1
| Plate | Marker | # Colonies |
| P104a + RBS | Kan | 0 |
| P104a + BIG | Kan | 0 |
| (+) Ctrl (puc19) | Amp | 200 |
| (-) Ctrl (EB buffer) | Kan | 0 |
Re-doing Cloning with P75, P63, cI, and P104a 7/31
PCR of PGW7 to get cI 7/31
| ' | cI857 BIG | cI857 RBS |
| Fwd Primer | 2 uL | 2 uL |
| Reverse Primer | 2 uL | 2 uL |
| PCR Supermix | 90 uL | 90 uL |
| Template | 2 uL | 2 uL |
| Water | 4 uL | 4 uL |
| Total | 100 uL | 100 uL |
Re-digestion of P75, P63, cI, and P104a 7/31
| ' | E1 P63b 07 | E1 P75a | E1 P75b | P104a |
| DNA | 30 uL | 10 uL | 10 uL | 9 uL |
| 100x BSA | 0.5 uL | 0.25 uL | 0.25 uL | 0.25 uL |
| 10x Buffer 2 | 5 uL | 2.5 uL | 2.5 uL | 2.5 uL |
| EcoRI | 1 uL | 2 uL | 3 uL | 4 uL |
| RE 2 | 1 uL SpeI | 1 uL XbaI | 1 uL XbaI | 1 uL XbaI |
| Water | 12.5 uL | 10.25 uL | 10.25 uL | 10.25 uL |
| Volume | 50 uL | 25 uL | 25 uL | 25 uL |
Gels from Digestions 8/1
Gels of Ligation Reactions 7/31
Ligation of P75 and P63 8/1
| ' | E1 P75a + P63b | E1 P75b + P63b | E1 P75a + P63b | E1 P75b + P63b | E1 P75a + P63b | E1 P75b + P63b |
| Insert | 6 uL | 6 uL | 5 uL | 5 uL | 1 uL | 1 uL |
| Vector | 2 uL | 2 uL | 1 uL | 1 uL | 1 uL | 1 uL |
| Dilution Buffer | 2 uL | 2 uL | 2 uL | 2 uL | 2 uL | 2 uL |
| Water | 0 uL | 0 uL | 2 uL | 2 uL | 6 uL | 6 uL |
| Total | 10 uL | 10 uL | 10 uL | 10 uL | 10 uL | 10 uL |
| Then add: | ||||||
| Ligation Buffer | 10 uL | 10 uL | 10 uL | 10 uL | 10 uL | 10 uL |
| Ligase | 1 uL | 1 uL | 1 uL | 1 uL | 1 uL | 1 uL |
| Total | 21 uL | 21 uL | 21 uL | 21 uL | 21 uL | 21 uL |
Transformed in E1.
Plates from Transformations 8/2
| Plate | Marker | # Colonies | Description |
| E1 P75a + P63b | Kan | ||
| E1 P75b + P63b | Kan | ||
| E1 P75a + P63b | Kan | ||
| E1 P75b + P63b | Kan | ||
| E1 P75a + P63b | Kan | ||
| E1 P75b + P63b | Kan | ||
| puc19 + ctrl | Amp | ||
| (-) ctrl | Kan |
Western Blot to Test IPTG and Heat Induction of Lac Systems
Induction and Preparing Lysate 7/29
| Sample | Protein Concentration |
| E1 P41 30 | 3.538645418 |
| E1 P41 30+IPTG | 3.397609562 |
| E1 P41 40 | 5.171713147 |
| E1 P84 30 | 3.413944223 |
| E1 P84 30+IPTG | 3.468924303 |
| E1 P84 40 | 4.507171315 |
| E1 P85 30 | 3.565338645 |
| E1 P85 30+IPTG | 3.680478088 |
| E1 P85 40 | 4.986055777 |
| E1 P30 30 | 3.359760956 |
| E1 P30 30 + IPTG | 0.502390438 |
| E1 P30 40 | 4.819123506 |
| Nat Cells 30 | 4.076494024 |
| Nat Cells OLD iptg | 4.479681275 |
| Nat Cells NEW IPTG | 4.276494024 |
| S1 p59B + p13 30 | 7.051394422 |
| S1 p59B + p13 IPTG | 5.019123506 |
| S1 P13 + P59b 1 30 | 6.422709163 |
| S1 P13 + P59b 1 IPTG | 5.163346614 |
| S1 P13 + P59b 2 30 | 6.125896414 |
| S1 P13 + P59b 2 IPTG | 4.337848606 |
| S1 P27 + P59b 2 30 | 2.854581673 |
| S1 P27 + P59b 2 IPTG | 4.25059761 |
| S1 P59b 30 | 6.311553785 |
| S1 P59b IPTG | 5.702788845 |
| S1 P13 (-) 30 | 5.293227092 |
| S1 P13 (-) IPTG | 6.001195219 |
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 | 50 | Small, with pink centers. Also some clusters of smaller colonies-- didn\'t pick any of those. |
| E1 P3+mtrB notBB B | Kan | 50 | Small, with pink centers. Also some clusters of smaller colonies-- didn\'t pick any of those. |
| E2 P1+mtrB A | Kan | 50 | Small, with pink centers. Also some clusters of smaller colonies-- didn\'t pick any of those. |
| E2 P1+mtrB B | Kan | 50 | Small, with pink centers. Also some clusters of smaller colonies-- didn\'t pick any of those. |
| E2 P3+mtrB NotBB A | Kan | 50 | Small, with pink centers. Also some clusters of smaller colonies-- didn\'t pick any of those. |
| E2 P3+mtrB NotBB B | Kan | 50 | Small, with pink centers. Also some clusters of smaller colonies-- didn\'t pick any of those. |
| (+) Ctrl (P59b) | Kan | 100s | Small, with pink centers. Also some clusters of smaller colonies. |
| (-) Ctrl (EB Buffer) | Kan | 0 |
Restreaked plates and picked two colonies per plate for liquid cultures and glycerol stocks.
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)
Ligation with promoters
We attempted ligations of P38 and P39 with the proper sized P17s excised from the gels in a 2:6 ratio with TOP10 cells. No plates (Kan) had colonies.
Mutagenesis of Cph1 07/29
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 P87B in TOP10 as well.
Transformation results 07/30
We used the TOP10 transformation protocol for both cell types. We plated 125 μL of each sample on two plates.
| Strain | Plate | DNA | # colonies (plate1, plate2) | Amount of DNA (μL) |
| E2 | Cm | 87A mut | ~400 each | 4 |
| E2 | Cm | 87B mut | TMTC, TMTC | 4 |
| E5 | Carb | PCR + control from kit | 160, 128 | 1 |
| E5 | Carb | pUC18 | TMTC, TMTC | 1 |
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.
P3A+(E1 P39+51 C) didn't grow in liquid culture.
RE digests 07/30
Lane 1: 1 kb ladder
Lane 2: P3A + (E1 P38 +51 D) uncut (3155)
Lane 3: P3A + (E1 P38 +51 D) cut SP (3155)
Lane 4: P3A + (E1 P38 +51 C) cut SP (3155)
Lane 5: P3A + (E1 P38 +51 B) cut SP (3155)
Lane 6: P3A + (E1 P39 +51 A) uncut (3155)
Lane 7: P3A + (E1 P39 +51 A) cut SP (3155)
Lane 8: P3A + (E1 P39 +51 B) cut SP (3155)
Lane 9: P3A + (E1 P39 +51 C) cut SP (3155)
Lane 10: P3A + (E1 P39 +51 D) cut SP (3155)
Lane 11: E1 P63+98 B uncut
Lane 12: E1 P63+98 B cut XP (~2200)
Lane 13: E1 P63+98 A cut XP (~2200)
Lane 14: E2 P63+98 B cut XP (~2200)
Lane 15: E2 P63+98 A cut XP (~2200)
.
RE digests 07/31
gel 1
Lane 1: 1 kb ladder
Lane 2: E5P105D cut ApaLI and PstI
Lane 3: E2P105A cut ApaLI and PstI
Lane 4: E5P105F cut ApaLI and PstI
Lane 5: E5P105G cut ApaLI and PstI
Lane 6: E2P105E cut ApaLI and PstI
Lane 7: E5P105B cut ApaLI and PstI
Lane 8: E5P105H cut ApaLI and PstI
Lane 9: E2P105E cut ApaLI and PstI
Lane 10: E5P105A cut ApaLI and PstI
Lane 11: E2P105B cut ApaLI and PstI
Lane 12: E2P105B uncut
gel 2
Lane 2: 1 kb ladder
Lane 3: E2P105D uncut
Lane 4: E5P105C cut ApaLI and PstI
Lane 5: E2P105C cut ApaLI and PstI
Lane 6: E2P105E cut ApaLI and PstI
Lane 7: P87A uncut
Lane 8: P87A cut ApaLI and PstI
Lane 9: P87B cut ApaLI and PstI
Lane 10: E1P63+98 E uncut
Lane 11: E1P63+98 E cut XP
Lane 12: E2P63+98 E cut XP
Lane 13: E2P63+98 F cut XP
Lane 14: E2P63+98 F cut XP
Western Blot
Testing ts-lac inducible system
MXH PCR 08/01
