Difference between revisions of "IGEM:Harvard/2008/Lab Notebooks/DailyBook/Week7/Chemical and Light"

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(Plate Results 8/6)
(Digestion and Ligation of P63 + cI857 (Both Primer Sets))
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==Digestion and Ligation of P63 + cI857 (Both Primer Sets)==
 
==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===
 
===Digestion of cI857 PCR product and P63 8/5===
 +
 +
{| {{table}}
 +
| align="center" style="background:#f0f0f0;"|''''''
 +
| align="center" style="background:#f0f0f0;"|'''P63 EX'''
 +
| align="center" style="background:#f0f0f0;"|'''cI857 RBS (GP) ES'''
 +
| align="center" style="background:#f0f0f0;"|'''cI857 BIG (GP) ES'''
 +
| align="center" style="background:#f0f0f0;"|'''cI857 RBS (PP) ES'''
 +
| align="center" style="background:#f0f0f0;"|'''cI857 BIG (PP) ES'''
 +
| align="center" style="background:#f0f0f0;"|'''P104 EX'''
 +
|-
 +
| DNA||10 uL||3 uL||7 uL||10 uL||10 uL||10 uL
 +
|-
 +
| 100X BSA||0.25 uL||0.25 uL||0.25 uL||0.25 uL||0.25 uL||0.25 uL
 +
|-
 +
| 10X Buffer 2||2.5 uL||2.5 uL||2.5 uL||2.5 uL||2.5 uL||2.5 uL
 +
|-
 +
| EcoRI||1 uL||1 uL||1 uL||1 uL||1 uL||1 uL
 +
|-
 +
| Enzyme 2||1 uL XbaI||1 uL SpeI||1 uL SpeI||1 uL SpeI||1 uL SpeI||1 uL XbaI
 +
|-
 +
| Water||10.25 uL||17.25 uL||13.25 uL||10.25 uL||10.25 uL||10.25 uL
 +
|-
 +
| Volume||25 uL||25 uL||25 uL||25 uL||25 uL||25 uL
 +
|}
 +
 +
Christina digested the same samples of P63, cI857 RBS GP and PP, and cI857 BIG GP and PP with EcoRI buffer. P104 was cut 8/6 in the morning.
 +
 +
====Gel results 8/6====
 +
{| {{table}}
 +
!rowspan=7|[[Image:8-7-08_p104and63_digest_al.jpg]]
 +
!colspan=2 align="center" style="background:#f0f0f0;"|'''1% agarose gel visualized using EtBr/UV'''
 +
|-
 +
| align="center" style="background:#f0f0f0;"|'''Lane'''
 +
| align="center" style="background:#f0f0f0;"|'''Sample'''
 +
|-
 +
| 1||1 kB Ladder
 +
|-
 +
| 2||P63
 +
|-
 +
| 3||P63c
 +
|-
 +
| 4||P104
 +
|-
 +
| 5||1 kB Ladder
 +
|}
 +
 
=mtrB, P97, P63 digests 08/06=
 
=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).
 
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).

Revision as of 15:33, 10 August 2008

PCR

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

8-04 gel 1 MXHTA.jpg 1% agarose gel visualized using EtBr/UV
Lane Contents (approx. expected band size)
1 1 kb plus ladder
2-5 cph-EnvZ 50 °C annealing temperature (2.3kb)
6-7 P1 (3kb)
8-9 P3 (3kb)
10-11 mtrB (2.1kb)
12-13 HO-pcyA (1.4kb)
14-15 ompR (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
8-04 gel 2 MXHTA.jpg 8-04 gel 3 MXHTA.jpg 8-04 gel 4 MXHTA.jpg
P98+63 Mix
8-04 gel 5 MXHTA.jpg 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

08-05 cph p1,3 mtrb mxh.jpg 1.2% agarose E-gel visualized using EtBr/UV
Lane Contents (approx. expected band size)
1-8 P105 (50.8-58.3 annealing temp, increases LTR) - 2.2kb
9 1kb plus ladder
10 P1 not BB - 3kb
11 P3 not BB - 3kb
12 mtrB not BB - 2.1kb
8-5 cph p1,3 hodgepodge.jpg Gel for excision (includes old PCR samples)
Lane Contents (approx. expected band size)
1, 15 1kb plus ladder
2-3 P105 PCR product
4-6 P3 PCR product
7-9 mtrB not BB PCR product
10 P1 PCR product
11-12 ompR PCR product (720bp)
13 HO-pcyA (1.4kb)
14 CDF 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) Kan 2 No fluor
XL10-Gold P75b + P63b (1:1) Kan 6 1 fluor
XL10-Gold P75a + P63b (6:2) Kan 0
XL10-Gold P75b + P63b (6:2) Kan 1 fluor
XL10-Gold P75a + P63b (7:1) Kan 1 no fluor
XL10-Gold P75b + P63b (7:1) Kan 2 both fluor
XL10-Gold Dephos P75a Kan 1
XL10-Gold Dephos p75b Kan 0
XL10-Gold P3 + (P39+p51) 2/6 Kan
XL10-Gold P3 Topo w/ XGAL Amp
XL10-Gold P1 Topo w/ XGAL Amp
XL10-Gold mtrB Topo w/ XGAL Amp
XL10-Gold pUC18 Amp 100s White, very small
XL10-Gold negative control Amp many extremely small
XL10-Gold negative control Kan 0

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.

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

9-19

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

20-30

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

31-41

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

42-52

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

53-63

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

64-74

8-6 pcr third 11 from back.jpg 1% agarose gel visualized using EtBr/UV
Lane Contents (approx. expected band size)
1-11 ompR + P1 ligation colony PCR (1kb)
12 1 kb plus ladder

75-85

8-6 pcr second 11 from back.jpg 1% agarose gel visualized using EtBr/UV
Lane Contents (approx. expected band size)
1-2 ompR + P1 ligation colony PCR (1kb)
3-11 ompR + P3 ligation colony PCR (1kb)
12 1 kb plus ladder

86-94, P20 controls

8-6 colony pcr mxh last 11.jpg 1% agarose gel visualized using EtBr/UV
Lane Contents (approx. expected band size)
1-9 ompR + P3 ligation colony PCR (1kb)
10 P20 colony PCR (1122)
11 P20 plasmid PCR
12 1 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) 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

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

8-5-08 analydig1 al.jpg 1% agarose gel visualized using EtBr/UV
Lane Sample
1 1 kB Ladder
2 E1 RBS 4R, 2D 1
3 E1 RBS 4R, 2D 2
4 E1 RBS 4R, 2D 3
5 E1 RBS 4R, 2D 4
6 E1 BIG 4R, 2D 1
7 E1 BIG 4R, 2D 2
8 E1 BIG 4R, 2D 3
9 E1 BIG 4R, 2D 4
10 E1 RBS 2R, 2D Xg 1
11 E1 RBS 2R, 2D Xg 2
12 E1 RBS 2R, 2D Xg 3


8-5-08 analydig2 al.jpg 1% agarose gel visualized using EtBr/UV
Lane Sample
1 BIG 4R, 4D Xg 1
2 BIG 4R, 4D Xg 2
3 BIG 4R, 4D Xg 3
4 mtrB 0.5 A
5 mtrB 0.5 B
6 mtrB 2 A
7 mtrB 2 B
8 RBS gel pur 2 A
9 RBS gel pur 2 B
10 BIG gel pur 2 A
11 BIG gel pur 2 B
12 1kB 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
DNA 30 uL 6 uL 11 uL
100x BSA 0.5 uL 0.25 uL 0.25 uL
10x Buffer 5 uL Buffer 2 2.5 uL Buffer 2 2.5 uL Buffer 2
RE 1 EcoRI 1 uL EcoRI 1 uL EcoRI 1 uL
RE 2 SpeI 1 uL XbaI 1 uL XbaI 1 uL
Water 12.5 uL 14.25 uL 9.25 uL
Volume 50 uL 25 uL 25 uL


Gel of Digestion 8/5

8-5-08 testlig al.jpg 1% agarose gel visualized using EtBr/UV
Lane Sample
1 1 kB Ladder
2 Christina\'s Vector (CE)
3 Christina\'s Insert (CI)
4 Christina\'s Vector (IE)
5 Christina\'s Insert (IE)
6 P75 EX
7 P63 ES
8 P18 EX

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

Used dephosphorylated and undephosphorylated vector.

Plate Results 8/6

Plate # Colonies Notes
pUC19 Amp I 31
Negative controls: . .
AmpI just cells 0
AmpI ligC dephos. IE vector only 8
AmpI ligC Ephos IE vector only v+i 1
AmpI ligC G8 SP just vector 6
AmpC ligC G8 SP just vector 7
AmpC ligC dephos IE vector only 5
DNA Positive controls: .
AmpC ligC G8 + G10 >200
AmpI ligC G8 + G10 81
AmpI ligI G8 + G10 78
AmpC ligI G8 + G10 127
whole thing: .
AmpI ligC CE CG 103
AmpI ligC CE dephos CG 45
AmpC ligC CE dephos 57
AmpC ligC CE CG 224
uh oh: .
AmpI ligC IE CG 3
AmpC ligC IE CG 7
AmpC ligC IE dephos V + I 46
iGEM DNA .
AmpI (-) 3
AmpI (- Dephos) 0
Kan 63+75 64
Kan 63 + 75 Dephos 0
AmpI 63+ 18 100+
AmpI 63+18 dephos 40

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

Digestion of cI857 PCR product and P63 8/5

' P63 EX cI857 RBS (GP) ES cI857 BIG (GP) ES cI857 RBS (PP) ES cI857 BIG (PP) ES P104 EX
DNA 10 uL 3 uL 7 uL 10 uL 10 uL 10 uL
100X BSA 0.25 uL 0.25 uL 0.25 uL 0.25 uL 0.25 uL 0.25 uL
10X Buffer 2 2.5 uL 2.5 uL 2.5 uL 2.5 uL 2.5 uL 2.5 uL
EcoRI 1 uL 1 uL 1 uL 1 uL 1 uL 1 uL
Enzyme 2 1 uL XbaI 1 uL SpeI 1 uL SpeI 1 uL SpeI 1 uL SpeI 1 uL XbaI
Water 10.25 uL 17.25 uL 13.25 uL 10.25 uL 10.25 uL 10.25 uL
Volume 25 uL 25 uL 25 uL 25 uL 25 uL 25 uL

Christina digested the same samples of P63, cI857 RBS GP and PP, and cI857 BIG GP and PP with EcoRI buffer. P104 was cut 8/6 in the morning.

Gel results 8/6

8-7-08 p104and63 digest al.jpg 1% agarose gel visualized using EtBr/UV
Lane Sample
1 1 kB Ladder
2 P63
3 P63c
4 P104
5 1 kB Ladder

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 old 10 min 2:6 DH5-alpha 1
mtrB not BB+P1 not BB 10 min 2:6 DH5-alpha 4
mtrB not BB+P1 not BB 5 min 2:6 TOP10 88
mtrB not BB+P3 not BB old 10 min 2:6 DH5-alpha 0
mtrB not BB+P3 not BB 10 min 2:6 DH5-alpha 8
mtrB not BB+P3 not BB 5 min 2:6 TOP10 40
mtrB+P63 10 min 2:6 TOP10 48
mtrB+P63 10 min 1:7 DH5-alpha 5
mtrB+P97 10 min 2:6 DH5-alpha TMTC
mtrB+P97 10 min 1:7 DH5-alpha 200
P105+P1 not BB old 10 min 2:6 DH5-alpha 5
P105+P1 not BB 10 min 2:6 DH5-alpha 1
P105+P1 not BB 5 min 2:6 TOP10 2
P105+P3 not BB old 10 min 2:6 DH5-alpha 1
P105+P3 not BB 10 min 2:6 DH5-alpha 8
P105+P3 not BB 5 min 2:6 TOP10 55

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

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)

2.png

None appear to be correct.

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

3.png

27, 28 picked.

RE digests 08/09

8-10 gel 1 MXHTA.jpg

Lane 1: 1 kb ladder

Lane 2: mtrB+63 #28 cut XP (~2200+3.1kb)

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

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

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

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

4.png

51 picked.

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

5.png

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

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

6.png

82, 87, 95 picked.

RE digests 08/07

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-alpha 2 (vector)!6 (water) 0
P45+108 7 ul 100 ul DH5-alpha 2:6 1
P17 in p15A (vector control) 7 ul 100 ul DH5-alpha 1 (vector):7 (water) 188
(P17 in p15A)+P39 7 ul 50 ul TOP10 1:7 1
(P17 in p15A)+P39 7 ul 100 ul DH5-alpha 1:7 0
(P17 in p15A)+P38 7 ul 50 ul TOP10 1:7 3
(P17 in p15A)+P38 7 ul 100 ul DH5-alpha 1:7 0

Colony PCR 8/9

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

08-09 colony mxh.jpg 1.2% E-gel visualized using EtBr/UV
Lane Contents (approx. expected band size)
1 P39+P17 in P1/3- col#1 (~940bp)
2-5 P38+P17 in P1/3- col#2-5 (~940bp)
6 1kb ladder
8-9 P108+P45 (same sample) (~2.3kb)
10 ligation control (no insert)

Since the bands are at least plausible, the P38/39 in P1/3 samples were grown up and miniprepped.

Retransformation 8/9

We retransformed 5μL of the P39+17 in P1/3 ligation into 50μL DH5α and obtained two colonies.

Colony PCR shows that one of these does not contain the right construct. The other was set up in 5ml culture.

08-10 colpcr ta.jpg

1kb ladder, colony A, colony B (both should have ~950 products if correct)

New ligations and 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.

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.