IGEM:Harvard/2008/Lab Notebooks/DailyBook/Week7/Chemical and Light

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(RE digests 08/09)
(Analytical Digest Gels from TOPO Cloning 8/5)
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==Analytical Digest Gels from TOPO Cloning 8/5==
==Analytical Digest Gels from TOPO Cloning 8/5==
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[Gels here]
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{| {{table}}
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!rowspan=14|[[Image:8-5-08_analydig1_al.jpg]]
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!colspan=2 align="center" style="background:#f0f0f0;"|'''1% agarose gel visualized using EtBr/UV'''
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|-
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| align="center" style="background:#f0f0f0;"|'''Lane'''
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| align="center" style="background:#f0f0f0;"|'''Sample'''
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|-
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| 1||1 kB Ladder
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|-
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| 2||E1 RBS 4R, 2D 1
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|-
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| 3||E1 RBS 4R, 2D 2
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|-
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| 4||E1 RBS 4R, 2D 3
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|-
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| 5||E1 RBS 4R, 2D 4
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|-
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| 6||E1 BIG 4R, 2D 1
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|-
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| 7||E1 BIG 4R, 2D 2
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|-
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| 8||E1 BIG 4R, 2D 3
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|-
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| 9||E1 BIG 4R, 2D 4
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|-
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| 10||E1 RBS 2R, 2D Xg 1
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|-
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| 11||E1 RBS 2R, 2D Xg 2
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|-
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| 12||E1 RBS 2R, 2D Xg 3
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|}
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Strange bands, but sent several clones for sequencing anyway.
+
 
 +
{| {{table}}
 +
!rowspan=14|[[Image:8-5-08_analydig2_al.jpg]]
 +
!colspan=2 align="center" style="background:#f0f0f0;"|'''1% agarose gel visualized using EtBr/UV'''
 +
|-
 +
| align="center" style="background:#f0f0f0;"|'''Lane'''
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| align="center" style="background:#f0f0f0;"|'''Sample'''
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|-
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| 1||BIG 4R, 4D Xg 1
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|-
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| 2||BIG 4R, 4D Xg 2
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|-
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| 3||BIG 4R, 4D Xg 3
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|-
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| 4||mtrB 0.5 A
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|-
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| 5||mtrB 0.5 B
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|-
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| 6||mtrB 2 A
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|-
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| 7||mtrB 2 B
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|-
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| 8||RBS gel pur 2 A
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|-
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| 9||RBS gel pur 2 B
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|-
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| 10||BIG gel pur 2 A
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|-
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| 11||BIG gel pur 2 B
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|-
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| 12||1kB Ladder
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|}
 +
 
 +
Strange bands, but sent several clones for sequencing anyway.
==Digestion and Ligation of P18+P63 and P75+P63==
==Digestion and Ligation of P18+P63 and P75+P63==

Revision as of 16:07, 10 August 2008

Contents

PCR

8/4: Cph/EnvZ, P1, P3, mtrB not BB

Image:8-04_gel 1_MXHTA.jpg 1% agarose gel visualized using EtBr/UV
Lane Contents (approx. expected band size)
11 kb plus ladder
2-5cph-EnvZ 50 °C annealing temperature (2.3kb)
6-7P1 (3kb)
8-9P3 (3kb)
10-11mtrB (2.1kb)
12-13HO-pcyA (1.4kb)
14-15ompR (750bp)

HO-pcyA and ompR bands excised, purified, and digested (ApaLI & Kpn overnight).

8/4 Colony PCR of putative mtrB samples

We picked 24 colonies each of old putative P98 and newly transformed putative P98+63 for colony PCR using the BioBrick primers and 55°C annealing for 30 cycles. If the colonies contained mtrB, a band at ~2.1 kb should appear. No colonies appeared to contain mtrB.

All gels with 1kb plus ladder

P98 P98 P98+63
Image:8-04_gel 2_MXHTA.jpgImage:8-04_gel 3_MXHTA.jpgImage:8-04_gel 4_MXHTA.jpg
P98+63 Mix
Image:8-04_gel 5_MXHTA.jpgImage:8-04_gel 6_MXHTA.jpg

Lane 1: 1 kb plus ladder
Lane 2: P98+63 (~2100)
Lane 3: P98+63 (~2100)
Lane 4: P98 (~2100)
Lane 5: P98 (~2100)

8/4 CphEnvZ, P1, P3, mtrB

Pl05 (CphEnvZ w/ PstI site mutated out) conditions: 5min @ 94°C → 40x [30s @ 94°C → 30s @ (50.8, 51.5, 52.6, 53.9, 55.4, 56.7, 57.7, 58.3)°C → 2:30 @ 72°C] → 7min @ 72°C → ∞ @ 4°C

8/5: gels

Image:08-05_cph_p1,3_mtrb_mxh.jpg 1.2% agarose E-gel visualized using EtBr/UV
Lane Contents (approx. expected band size)
1-8P105 (50.8-58.3 annealing temp, increases LTR) - 2.2kb
91kb plus ladder
10P1 not BB - 3kb
11P3 not BB - 3kb
12mtrB not BB - 2.1kb
Image:8-5_cph_p1,3_hodgepodge.jpg Gel for excision (includes old PCR samples)
Lane Contents (approx. expected band size)
1, 151kb plus ladder
2-3P105 PCR product
4-6P3 PCR product
7-9mtrB not BB PCR product
10P1 PCR product
11-12ompR PCR product (720bp)
13HO-pcyA (1.4kb)
14CDF PCR product (900bp)


Bands excised and digested: Cph/P105 (AK), P3 (AK), mtrB (XP, ES)

Bands excised and saved: ompR, HO-pcyA, CDF

Ligations and transformations 08/06

We ligated P105 and P1/P3 not BB, and mtrB not BB and P1/P3 not BB. We tried letting the ligation reactions run for 5 and 10 minutes. We used P1/P3 not BB from 08/05 and from another date (older). We transformed using TOP10 and DH5α. We also retransformed P108, the Lac QPI with a high constitutive promoter in a p15A vector.

Results below

TOPO cloning

08/04

P1, mtrB

These had a few white colonies. Colony PCR, however, indicated that the correct PCR product was not incorporated.

ompR

All colonies were blue.

HO-pcyA

No colonies.

Transforming in XL10-Gold cells

Plate Marker # Colonies Description
XL10-Gold P75a + P63b (1:1)Kan2No fluor
XL10-Gold P75b + P63b (1:1)Kan61 fluor
XL10-Gold P75a + P63b (6:2)Kan0
XL10-Gold P75b + P63b (6:2)Kan1fluor
XL10-Gold P75a + P63b (7:1)Kan1no fluor
XL10-Gold P75b + P63b (7:1)Kan2both fluor
XL10-Gold Dephos P75a Kan1
XL10-Gold Dephos p75b Kan0
XL10-Gold P3 + (P39+p51) 2/6Kan
XL10-Gold P3 Topo w/ XGALAmp
XL10-Gold P1 Topo w/ XGALAmp
XL10-Gold mtrB Topo w/ XGALAmp
XL10-Gold pUC18Amp100sWhite, very small
XL10-Gold negative control Ampmanyextremely small
XL10-Gold negative controlKan0

Ligations

HO-pcyA, ompR, CDF

All three ligations yielded >50 colonies on the TOP10 cell plate with a 5 minute ligation in all combinations (HO-pcyA and ompR with P1 and old P3; CDF with P3). The P90 (CDF+P3) did not fluoresce. The DH5α cells did not have nearly as many (<5) and ligation times (10min, 30 min) did not appear to be significant.

Colony PCR

We picked at least 18 colonies from each ligation set, patched a master plate, inoculated liquid cultures, and performed 12.5 μL colony PCR reactions. The BBp/sfx primer pair was used, annealing temperature was 50°C for first 10 cycles and 54°C for rest of the cycles. 2 controls were performed: one using a picked P20 colony, and another with miniprepped P20 plasmid.

Digests, 1-8

We ran the digests to see if PCR mutations could have introduced an internal cut site. However, even with the small amount of purified digest we had left, we could see a properly sized band for each of P1, ompR, and HO-pcyA PCR products.

Image:8-6_pcr_seveneth_11fromback.jpg 1% agarose gel visualized using EtBr/UV
Lane Contents (approx. expected band size)
11 kb plus ladder
2Old P1 PCR product AK digested (3kb)
3ompR PCR product AK digested (720bp)
4HO-pcyA PCR product AK digested (1.4kb)
5-12HO-pcyA + P1 ligation colony PCR (1.6kb)

9-19

Image:8-6_pcr_eighteth_11fromback.jpg 1% agarose gel visualized using EtBr/UV
Lane Contents (approx. expected band size)
11 kb plus ladder
2-11HO-pcyA + P1 ligation colony PCR (1.6kb)
12HO-pcyA + P3 ligation colony PCR (1.6kb)

20-30

Image:8-6_pcr_nineth_11 from back.jpg 1% agarose gel visualized using EtBr/UV
Lane Contents (approx. expected band size)
11 kb plus ladder
2-12HO-pcyA + P3 ligation colony PCR (1.6kb)

31-41

Image:8-6_pcr_sixth_11_from_back.jpg 1% agarose gel visualized using EtBr/UV
Lane Contents (approx. expected band size)
11 kb plus ladder
2-7HO-pcyA + P3 ligation colony PCR (1.6kb)
8-12CDF + P3 ligation colony PCR (900bp)

42-52

Image:8-6_pcr_fifth_11_from_back.jpg 1% agarose gel visualized using EtBr/UV
Lane Contents (approx. expected band size)
11 kb plus ladder
2-12CDF + P3 ligation colony PCR (900bp)

53-63

Image:8-6_pcr_fourth_11_from_back.jpg 1% agarose gel visualized using EtBr/UV
Lane Contents (approx. expected band size)
11 kb plus ladder
2-7CDF + P3 ligation colony PCR (900bp)
8-12ompR + P1 ligation colony PCR (1kb)

64-74

Image:8-6_pcr_third_11_from_back.jpg 1% agarose gel visualized using EtBr/UV
Lane Contents (approx. expected band size)
1-11ompR + P1 ligation colony PCR (1kb)
121 kb plus ladder

75-85

Image:8-6_pcr_second_11_from_back.jpg 1% agarose gel visualized using EtBr/UV
Lane Contents (approx. expected band size)
1-2ompR + P1 ligation colony PCR (1kb)
3-11ompR + P3 ligation colony PCR (1kb)
121 kb plus ladder

86-94, P20 controls

Image:8-6_colony_pcr_mxh_last_11.jpg 1% agarose gel visualized using EtBr/UV
Lane Contents (approx. expected band size)
1-9ompR + P3 ligation colony PCR (1kb)
10P20 colony PCR (1122)
11P20 plasmid PCR
121 kb plus ladder

Making Thermoinducible cI System

Ligation from 8/1

Plate Results (also listed in Week 6):

Plate Marker # Colonies Description
E1 P75a + P63b (1:1)Kan0
E1 P75b + P63b (1:1)Kan0
E1 P75a + P63b (6:2)Kan1No Fluor, 3mm
E1 P75b + P63b (6:2)Kan1Fluorescent, 1mm.
E1 P75a + P63b (7:1)Kan0
E1 P75b + P63b (7:1)Kan0
Dephos P75b Kan0

Fluorescent colony was picked for liq culture and restreaked. Miniprep sent for sequencing-- ligation confirmed by sequencing (8/5).

Analytical Digest Gels from TOPO Cloning 8/5

Image:8-5-08_analydig1_al.jpg 1% agarose gel visualized using EtBr/UV
Lane Sample
11 kB Ladder
2E1 RBS 4R, 2D 1
3E1 RBS 4R, 2D 2
4E1 RBS 4R, 2D 3
5E1 RBS 4R, 2D 4
6E1 BIG 4R, 2D 1
7E1 BIG 4R, 2D 2
8E1 BIG 4R, 2D 3
9E1 BIG 4R, 2D 4
10E1 RBS 2R, 2D Xg 1
11E1 RBS 2R, 2D Xg 2
12E1 RBS 2R, 2D Xg 3


Image:8-5-08_analydig2_al.jpg 1% agarose gel visualized using EtBr/UV
Lane Sample
1BIG 4R, 4D Xg 1
2BIG 4R, 4D Xg 2
3BIG 4R, 4D Xg 3
4mtrB 0.5 A
5mtrB 0.5 B
6mtrB 2 A
7mtrB 2 B
8RBS gel pur 2 A
9RBS gel pur 2 B
10BIG gel pur 2 A
11BIG gel pur 2 B
121kB Ladder

Strange bands, but sent several clones for sequencing anyway.

Digestion and Ligation of P18+P63 and P75+P63

Done in tandem and side by side with Christina (except for digestion).

Digestion of P18, P63, and P75 8/4

' P63 P75b P18
DNA30 uL6 uL11 uL
100x BSA0.5 uL0.25 uL0.25 uL
10x Buffer5 uL Buffer 22.5 uL Buffer 22.5 uL Buffer 2
RE 1EcoRI 1 uLEcoRI 1 uLEcoRI 1 uL
RE 2SpeI 1 uLXbaI 1 uLXbaI 1 uL
Water12.5 uL14.25 uL9.25 uL
Volume50 uL25 uL25 uL


Gel of Digestion 8/5

Ligation of P18+P63 and P75+P63 8/5

Used dephosphorylated and undephosphorylated vector.

Plate Results 8/6

Digestion and Ligation of P63 + cI857 (Both Primer Sets)

Done with gel purified and pcr purified cI857, and in tandem with Christina.

Digestion of cI857 PCR product and P63 8/5

mtrB, P97, P63 digests 08/06

We digested mtrB with ES and XP so that we can put it into the P63 (terminator) and P97 (RBS) vectors. We also digested P97 with SP (and dephosphorylated it).

Ligation transformation results 08/07

Plasmid Ligation time Ligation ratio (vector:insert) Strain No. of Colonies
mtrB not BB+P1 not BB old10 min2:6DH5-alpha1
mtrB not BB+P1 not BB10 min2:6DH5-alpha4
mtrB not BB+P1 not BB5 min2:6TOP1088
mtrB not BB+P3 not BB old10 min2:6DH5-alpha0
mtrB not BB+P3 not BB10 min2:6DH5-alpha8
mtrB not BB+P3 not BB5 min2:6TOP1040
mtrB+P6310 min2:6TOP1048
mtrB+P6310 min1:7DH5-alpha5
mtrB+P9710 min2:6DH5-alphaTMTC
mtrB+P9710 min1:7DH5-alpha200
P105+P1 not BB old10 min2:6DH5-alpha5
P105+P1 not BB10 min2:6DH5-alpha1
P105+P1 not BB5 min2:6TOP102
P105+P3 not BB old10 min2:6DH5-alpha1
P105+P3 not BB10 min2:6DH5-alpha8
P105+P3 not BB5 min2:6TOP1055

Colony PCR

Ladders (end of each block) alternate as low range (100, 200, 400, 800, 2000bp) and 1kb plus. Expected band sizes indicated.

1-16

Image:1.png

Lane 1: Plasmid control P85 (2.3kb)
Lane 2: Colony control P88 (2.2kb)
Lanes 3-16: P105+P3 ligation colony PCR (2.5kb)

4, 6, 14 look like they have potentially correct products, so liquid cultures were grown up.

17-24: P105+P1 ligation colony PCR (2.5kb)

Image:2.png

None appear to be correct.

25-48: mtrB+P63 ligation colony PCR (2.2kb)

Image:3.png

27, 28 picked.

49-64: mtrB+P97 ligation colony PCR (2.1kb)

Image:4.png

51 picked.

65-80: mtrB'+P3 ligation colony PCR (2.3kb)

Image:5.png

65, 69, 70, 78, 79, 80 picked.

81-96: mtrB'+P1 ligation colony PCR (2.3kb)

Image:6.png

82, 87, 95 picked.

RE digests 08/07

Image:8-08_gel_1_MXHTA.jpg

Lane 1: 1 kb ladder

Lane 2: E1P1+P17A cut EX (3652)

Lane 3: E1P1+P17C cut EX (3652)

Lane 4: E1P1+P17D cut EX (3652)

Lane 5: E2P1+P17A cut EX (3652)

Lane 6: E2P1+P17B cut EX (3652)

Lane 7: P108 cut SP (4155)

Lane 8: P45 cut XP (876; 2079)

Ligations 08/08

  • We ligated P17 in a p15A vector (P1 or P3) to P38 and P39. We used a vector to insert ratio of 1:7. We also prepared a ligation reaction with 1 μL vector (P17) and 7 μL water as a transformation control. We transformed 50 μL TOP10 cells with 7 μL DNA and 100 μL DH5α cells with 7 μL DNA. We also used 7 μL of the vector-only ligation to transform 7 μL DH5α cells.
  • To test the Lac QPI system, we ligated P45 (RBS+GFP+terminator) to P108. We used a vector to insert ratio of 2:6. We also prepared a ligation reaction with 1 μL vector (P108) and 7 μL water as a transformation control. We transformed 100 μL DH5α cells with 7 μL DNA and we also used 7 μL of the vector-only ligation to transform 7 μL DH5α cells.

Transformation results 08/09

Plasmid Amount of DNA Strain Ligation ratio (vector:insert by volume) Number of colonies
P108 (vector control)7 ul 100 ul DH5-alpha2 (vector)!6 (water)0
P45+1087 ul100 ul DH5-alpha2:61
P17 in p15A (vector control)7 ul100 ul DH5-alpha1 (vector):7 (water)188
(P17 in p15A)+P397 ul50 ul TOP101:71
(P17 in p15A)+P397 ul100 ul DH5-alpha1:70
(P17 in p15A)+P387 ul50 ul TOP101:73
(P17 in p15A)+P387 ul100 ul DH5-alpha1:70

Colony PCR 8/9

We picked all of the noncontrol colonies and one control colony for colony PCR using the BBp/sfx primers.

Image:08-09_colony_mxh.jpg 1.2% E-gel visualized using EtBr/UV
Lane Contents (approx. expected band size)
1P39+P17 in P1/3- col#1 (~940bp)
2-5P38+P17 in P1/3- col#2-5 (~940bp)
61kb ladder
8-9P108+P45 (same sample) (~2.3kb)
10ligation control (no insert)

Transforming LacZ parts

We attempted to transform the following parts: I732017, E0435, E0435, I732005 into our competent DH5α. E0435 (on CM) had 0 colonies). The others (on Carb) had 10-20 extremely small colonies (after 16+hrs of incubation) each. We picked 2 of each for liquid culture, but none grew.

We requested the parts directly from MIT.

RE digests 08/09

Image:8-10_gel_1_MXHTA.jpg

Lane 1: 1 kb ladder

Lane 2: mtrB+63 #28 cut XP (~2200)

Lane 3: E1P3A-P38+51C cut SP (4155)

Lane 4: E1P3A-P38+51D cut SP (4155)

Lane 5: E1P3A-P38+51B cut SP (4155)

Transformations 08/10

We retransformed our old P108+45 (2:6 vector to insert) ligation and the old vector control. We also did this ligation over with a 2:1 molecular ratio of insert to vector. Both P108 (4155 bp) and P45 (876 bp) were about 17 ng/μL. We used a ratio of 5.4:2.3 vector to insert by volume.

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