IGEM:Harvard/2008/Lab Notebooks/DailyBook/Week6/Chemical and Light

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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

7-30-08 p104a digest al.jpg 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
7-31-08 pcr.jpg 1% Agarose, visualized using EtBr/UV
Lane Sample
1 1 kB Ladder
2 RBS 1
3 RBS 2
4 100bp Ladder
5 BIG 1
6 BIG 2

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

8-1-08 digests amy.jpg 1% Agarose, visualized using EtBr/UV
Lane Sample
1 1 kB Ladder
2 E1 P75a
3 E1 P75b
4 100bp Ladder
5 E1 P104
6 E1 P63b

Gels of Ligation Reactions 7/31

7-31-08 egela.jpg 1% Agarose, visualized using EtBr/UV
Lane Sample
1 1 kb ladder
2 E1 P75b + P63a 6:2 7/25/08
3 E1 P75b + P63b 6:2 7/25/08
4 S1 P75a + P63a 6:2 7/25/08
5 S1 P75a + P63b 6:2 7/25/08
6 S1 P75b + P63a 6:2 7/25/08
7 S1 P75b + P63b 6:2 7/25/08
8 E1 P75b + P63a 5:1 7/25/08
9 E1 P75b + P63b 5:1 7/25/08
10 S1 P75a + P63a 5:1 7/25/08
11 S1 P75b + P63b 5:1 7/25/08
12 E1 P75b EX (Dephos) 7/25/08
7-31-08 egel b.jpg 1% Agarose, visualized using EtBr/UV
Lane Sample
1 100bp ladder
2 E1 P104a + RBS 6:2 7/30/08
3 E1 P104a + BIG 6:2 7/30/08
4 E1 P104a + RBS 5:1 7/30/08
5 E1 P104a + BIG 5:1 7/30/08
6 E1 P104a + RBS 6:2 7/31/08
7 E1 P104a + BIG 6:2 7/31/08
8 E1 P104a EX 7/30/08
9 P104 EX Dephos 7/30/08
10 RBS 7/29/08
11 BIG 7/29/08
12 1 kB Ladder

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

TOPO TA cloning of cI

TOPO TA cloning with gel extracted cI 8/1

' Volume
PCR Product 0.5 - 4 uL
Salt Solution 1 uL
TOPO Vector 1 uL
Water add to 6 uL

Transformed in E1.

Plate Results 8/2

Includes results for ligation of P75a and P75b with P63 (terminator). RBS indicates primer set RBS, and BIG indicated primer set BIG. These TOPO cloning reactions were done with gel purified cI857. Volumes of DNA in parentheses indicate the amount of DNA added to the cloning reaction (0.5, 2, or 4 uL).

Plate Marker # Colonies Description
E1 P75a + P63b (1:1) Kan 0
E1 P75b + P63b (1:1) Kan 0
E1 P75a + P63b (6:2) Kan 1 No Fluor, 3mm
E1 P75b + P63b (6:2) Kan 1 Fluorescent, 1mm.
E1 P75a + P63b (7:1) Kan 0
E1 P75b + P63b (7:1) Kan 0
Dephos P75b Kan 0
RBS (0.5 uL DNA) Amp 24 big, 5 small White, bi: 2mm. Smaller ones flanking larger colonies: <1 mm.
RBS (2 uL DNA) Amp ~40 big, 10-20 small White, big colonies: 2mm. Smaller ones flanking them: <1mm.
RBS (4 uL DNA) Amp ~50 big, 10-20 small. White, big colonies: 2mm. Smaller ones flanking them: <1mm.
BIG (0.5 uL DNA) Amp ~15 big White, big colonies: 2mm.
BIG (2 uL DNA) Amp ~25 big White, big colonies: 2mm.
BIG (4 uL DNA) Amp ~40 White, big colonies: 2mm.
mtrB BB (0.5 uL DNA) Amp ~40 White colonies, 2mm.
mtrB BB (2 uL DNA) Amp ~40-50 big, ~50 small White, big colonies: 2mm. Smaller ones flanking them: <1mm.
mtrB BB (4 uL DNA) Amp ~20 big, ~20 small White, big colonies: 2mm. Smaller ones in clusters: <1 mm.
puc19 + ctrl Amp 100 white, big colonies, 50+ small. White, big colonies: 2mm. Smaller ones in clusters: <1 mm.
(-) ctrl Kan 0
just TOPO vector Amp ~25 big White, big colonies: 1-2mm.
(-) ctrl Amp 0

PCR of PGW7 to get more cI 8/1

' 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

Gel of PCR 8/1

8-1-08 cIPCR al.jpg 1% Agarose, visualized using EtBr/UV
Lane Sample
1 1 kb Ladder
2 RBS
3 BIG
4 100bp ladder

Used in TOPO TA Cloning Reaction and transformation with E1.

TOPO TA with Fresh cI PCR Product 8/1

' RBS (0.5 uL DNA) RBS (2 uL DNA) RBS (4 uL DNA) BIG (0.5 uL DNA) BIG (2 uL DNA) BIG (4 uL DNA) Ctrl Template Just Vector
PCR Product 0.5 2 4 0.5 2 4 1 0
Salt Solution 1 1 1 1 1 1 1 1
TOPO Vector 1 1 1 1 1 1 1 1
Water 3.5 2 0 3.5 2 0 3 4

Transformed in E1-- added either 2 or 4 uL of cloning reaction. Had limited and suspect X-gal so only applied to some plates, and included X-gal control for contamination.

Plate Results 8/2

Includes results for control reactions for TOPO TA cloning system. RBS indicates primer set RBS, and BIG indicated primer set BIG. These TOPO cloning reactions were done with fresh PCR product of cI857 (confirmed with E-gel). Volumes of DNA in parentheses indicate the amount of DNA added to the cloning reaction (0.5, 2, or 4 uL). Volumes of DNA rxn indicates how much of the cloning reaction was added to the competent cells (either 2 or 4 uL). Presence or absence of X-gal indicated in Description.

Plate Marker # Colonies Description
RBS (0.5 uL DNA) 2 uL rxn Amp 100+ Had X Gal: 6 blue, rest white. All 1-2mm.
RBS (0.5 uL DNA) 4 uL rxn Amp 200+ No X Gal: all white. All 1mm.
RBS (2 uL DNA) 2 uL rxn Amp 100+ Had X Gal: ~25 blue, rest white. All 1-2 mm.
RBS (2 uL DNA) 4 uL rxn Amp 200+ No X Gal: all white. All 0.5-1 mm.
RBS (4 uL DNA) 2 uL rxn Amp 100-200 No X Gal: all white. All 0.5-1 mm.
RBS (4 uL DNA) 4 uL rxn Amp 100-200 big, ~5-10 tiny colonies Had X-Gal: 40-50 blue, rest white. Bigger colonies 0.5-1mm, tiny colonies 0.1-0.5mm, flanking big colonies.
BIG (0.5 uL DNA) 2 uL rxn Amp ~75 Had X-Gal: 23 blue, rest white. 0.5-1mm.
BIG (0.5 uL DNA) 4 uL rxn Amp 100+ No X Gal: all white. All 1mm.
BIG (2 uL DNA) 2 uL rxn Amp 100-200 No X-Gal: all white. All 0.5-1mm.
BIG (2 uL DNA) 4 uL rxn Amp 200+ Had X-gal:50-100 blue, rest white. All 0.5mm. Some are tinged blue.
BIG (4 uL DNA) 2 uL rxn Amp 100 No X-gal: All white, 1-2mm.
BIG (4 uL DNA) 4 uL rxn Amp 200 big, 25-50 tiny. Had X-Gal: 40-50 blue, rest white. Bigger colonies 0.5-1mm, tiny colonies 0.1-0.5mm, flanking big colonies.
Ctrl Template 2 uL rxn Amp 75-100 No X Gal: all white. All 0.5-1 mm.
Ctrl Template 4 uL rxn Amp 100 big, 25-50 tiny Had X-Gal: 25-50 blue, rest white. Bigger colonies 0.5-1mm, tiny colonies 0.1-0.5mm, flanking big colonies.
Just Vector 2 uL rxn Amp ~35 No X Gal: all white. All 1-2 mm.
Just Vector 4 uL rxn Amp 50+ Had X-Gal: 2 fully white--one on edge where X-gal probably not present, one closer to center. Rest are blue, but some, especially close to the edge, are much lighter blue. Would recommend not picking even colonies that are tinged blue and to try to not pick around blue colonies.
(+) puc 19 ctrl Amp ~100 All white, 1-2mm, not very evenly distributed.
(-) just cells Amp 0
(-) just X-gal Amp 0

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.

7-29 gel 1 MXHTA.jpg

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

7-30 gel 1 MXHTA.jpg

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

7-31 gel 1 MXHTA.jpg

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

7-31 gel 2 MXHTA.jpg

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

2008-03-18 2.jpg

Only the constitutive plasmids (p30 and p59b) and Natalie's plasmid when induced expressed the desired protein, indicating that neither the heat-inducible lac system nor the dual plasmid lac operon work.

Upper Gel: 1- S1 p13 and p59b 2 IPTG 2- S1 p13 and p59b 2 30°C 3- E1 p30 40°C 4- E1 p30 IPTG 5- E1 p30 30°C 6- E1 p85 40°C 7- E1 p85 IPTG 8- E1 p85 30°C 9- E1 p84 40°C 10- E1 p84 IPTG 11- E1 p84 30°C 12- Ladder 13- E1 p41 40°C 14- E1 p41 IPTG 15- E1 p41 30°C

Lower Gel: 1- pGEX (Natalie's cells) 30°C 2- Ladder 3- pGEX our IPTG 4- pGEX Silver Lab's IPTG 5- S1 p59b and p13 6- S1 p59b and p13 IPTG 7- S1 p13 and p59b 1 8- S1 p13 and p59b 1 IPTG 9- S1 p27 and p59b 10- S1 p27 and p59b IPTG 11- S1 p59b 12- S1 p59b IPTG 13- S1 p13 14- S1 p13 IPTG 15- Ladder

RE digests and ligations 08/02

Lane 1: 1 kb ladder

Lane 2: P3 cut XP, dephosphorylated (2750)

Lane 3: P39+51 cut XP (1405)

8-02 gel 1.1 MXHTA.jpg

8-02 gel 1.2 MXHTA.jpg

Gel extraction:

These bands were cut from the gel and melted at 65 °C. This was used in one set of ligations. Another set was done using our normal gel extraction protocol. We used ligation ratios of 2:6 and 3:5 of vector to insert. The ligations were transformed into DH5α cells.

Results

Both transformations involving the DNA extracted using the new gel extraction protocol failed (no colonies). The the ligation with a ratio of 2:6 of P3 to P39+51 did not give any colonies, but the 3:5 ligation plate had 2 colonies. Both of these colonies were picked and grown up in liquid cultures.

Cutting GFP out of P1

Plate Marker # of Colonies Description
tctr1 pUC19 (control) Carb ~125 scattered 1mm diameter colonies
P1 A (XS) 10 min Kan Too many to count ~0.5mm diameter colonies
P1 A (XS) 20 min Kan Too many to count ~0.5mm diameter colonies
P1 A (XS) 40 min Kan Too many to count ~0.5mm diameter colonies

ON RE digests and ligations 08/03

  • We cut P98 with ES to ligate it with a terminator, P63 cut with EX. This will give us RBS+mtrB+terminator. We also cut P98 with XmnI because the backbone and insert of P98 are around the same size, and digesting with XmnI cuts the backbone so it becomes smaller.
  • We also cut the QPIs (P17: Tet and P51: Lac) in a p15A vector with EX. We will add high and low constitutive promoters.

8-03 gel 1 MXHTA.jpg

Lane 1: 1 kb ladder

Lane 2: P98A uncut (~4200)

Lane 3: P98A cut ESXmnI (~2102; 1623, 433)

Lane 4: P98B cut ESXmnI (~2102; 1623, 433)

Lane 5: P98C cut ESXmnI (~2102; 1623, 433)

Lane 6: P98D cut ESXmnI (~2102; 1623, 433)

Lane 7: P98E cut ESXmnI (~2102; 1623, 433)

Lane 8: P98F cut ESXmnI (~2102; 1623, 433)

Lane 9: P63 cut EX (3284)

Lane 10: P3+51# cut EX (4120)

Lane 11: P1/3+51 cut EX (4120)

Lane 12: P3+51 cut EX (4120)

Lane 13: P3+17B uncut (3652)

Lane 14: P3+17 cut EX (~3652)

Lane 15: P3+17 cut EX (~3652)

Lane 16: 1 kb plus ladder

  • Since the P63 band in lane 9 looked like it could have also had uncut DNA, we used a sample of P63 from 07/16 in the ligations.
  • We used the standard QIAGEN gel extraction protocol.
  • We ligated using 2:6 of vector to insert. We let the P98+63 ligations run for 10, 20 and 30 minutes (the other ligations only ran for 10 minutes).
  • We transformed the P39+51, P39+17, P98+63 10' ligations into TOP10 cells. All of the other ligations were transformed into DH5α. We also transformed a positive control with pUC19.
  • Since there was no SOC medium left, we incubated in LB for 2 hours after heat shock.

Results

Plasmid Ligation time Number of colonies Cell type
pUC19 control N/A 221 DH5-alpha
P38+(P51 in P3) 10' 1 TOP10
P38+(P51 in P3) 20' 0 DH5-alpha
P38+(P51 in P3) 30' 5 DH5-alpha
P39+(P51 in P3) 10' 0 TOP10
P39+(P51 in P3) 20' 0 DH5-alpha
P39+(P51 in P3) 30' 1 DH5-alpha
P38+(P17 in P3) 10' 23 TOP10
P38+(P17 in P3) 20' 0 DH5-alpha
P38+(P17 in P3) 30' 0 DH5-alpha
P39+(P17 in P3) 10' 128 TOP10
P39+(P17 in P3) 20' 0 DH5-alpha
P39+(P17 in P3) 30' 0 DH5-alpha
P98+63 10' TMTC (most) TOP10
P98+63 20' TMTC DH5-alpha
P98+63 30' TMTC (least) DH5-alpha