IGEM:Harvard/2008/Lab Notebooks/DailyBook/Week8/Chemical and Light

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(Gel of Digestion 8/10)
(Ligation 8/11)
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| puc 19 (+) ctrl||Amp||0 colonies
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===Colony PCR of Transformations 8/12===
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{| {{table}}
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!rowspan=17|[[Image:8-13-08_colonypcr1_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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| 1||1 kb ladder
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| 2||E1 P104 + RBS TOPO 1 6:2
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| 3||E1 P63 + P18 (1) + RBS TOPO 1 A
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| 4||E1 P63 + P18 (1) + RBS TOPO 1 B
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| 5||E1 P63 + P18 (1) + RBS TOPO 3 A
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| 6||E1 P63 + P18 (1) + RBS TOPO 3 B
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| 7||E1 P63 + P18 (1) + RBS TOPO 5 A
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| 8||E1 P63 + P18 (2) + RBS TOPO 1 A
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| 9||E1 P63 + P18 (2) + RBS TOPO 1 B
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| 10||E1 P63 + P18 (2) + RBS TOPO 1 C
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| 11||E1 P63 + P18 (2) + RBS TOPO 3 A
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| 12||E1 P63 + P18 (2) + RBS TOPO 3 B
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|}
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{| {{table}}
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!rowspan=17|[[Image:8-13-08_colonypcr2_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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| 1||E1 P63 + P18 (2) + RBS TOPO 3 C
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| 2||E1 P63 + P18 (2) + RBS TOPO 3 D
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| 3||E1 P63 + P18 (2) + RBS TOPO 5 A
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| 4||E1 P63 + P18 (2) + RBS TOPO 5 B
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| 5||E1 P63 + P18 (2) + RBS TOPO 5 C
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| 6||1 kb Ladder
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| 7||
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| 8||
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Revision as of 10:43, 13 August 2008

Contents

MIT lacZ parts RE digests 08/11

Image:8-11_gel_1_MXHTA.jpg 1% agarose
Lane Table of Contents
11 kb ladder
2P109 MIT cut ES (cut out insert 3093; vector 2079)
3P110 MIT cut XP (insert 534; vector 3953)
4P111 MIT cut XP (insert 534; cut out vector 3755)
5P113 MIT cut XP (insert 534; vector 3973)
6P112 MIT cut XP (cut out insert 534; vector 3722)

P108+45 colony PCR, P1+mtrB test digest 8/11

Image:8-11_col_pcr_mxh.jpg 1.2% E-gel visualized using EtBr/UV
Lane Contents (approx. expected band size)
1-4P108+45 putative colony PCR #12-15 (2.3kb)
2P1+mtrB not BB test digest (ES) #82 (2.2+2.75kb)
3P1+mtrB not BB test digest (ES) #87 (2.2+2.75kb)
4P1+mtrB not BB test digest (ES) #95 (2.2+2.75kb)
5-121kb ladder

Nothing looks hopeful, so we'll wait until sequences come back.

Sequence results

All of the P1+mtrB not BBs aren't correct. 82 has not hits in BLAST, which 87 and 95 BLAST to pHELLSGATE- apparently some cloning vector.

Ligations/transformations 08/11

We ligated P109+63 to get RBS+full LacZ+terminator.

Plasmid Strain Number of colonies
P63 vector controlDH5-alpha0
P63+P109 (1:2 molar ratio)DH5-alpha17
P63+P109 (1:2 molar ratio)TOP10112

Colony PCR

We PCRed five colonies from each plate to check if they were the right size. We also used P109 as a positive control.

Image:8-12_gel_1_MXHTA.jpg

Lane 1: 1 kb plus ladder

Lane 2: P109 positive control (3093)

Lane 3: P109+63A (3188)

Lane 4: P109+63B (3188)

Lane 5: P109+63C (3188)

Lane 6: P109+63D (3188)

Lane 7: P109+63E (3188)

Lane 8: P109+63F (3188)

Lane 9: P109+63G (3188)

Lane 10: P109+63H (3188)

Lane 11: P109+63I (3188)

Lane 12: P109+63J (3188)

CDF

Alternative method

Using XbaI and NheI sites around the origin in pCDF, we can cut it out and ligate it into an XbaI digested P48. Depending on the orientation of the insert, the NheI-XbaI mixed site can be either just ahead of the Cm resistance (what we want) or after the E site in the BBMCS. This would destroy the prefix, so we'll use digest multiple colonies with XS for the correctly inserted origin-resistance part to put into the base pSB3K3.

pCDF, P48 digest results 8/12

The pCDF Xbal-NheI double digest (overnight, 25μL, 0.75μL of each enzyme) appeared fine, and the 800bp insert was gel purified.

P48 Xbal mysteriously yielded 2 bands, so is going to be repeated with another tube of P48.

ompR (P115) digests 8/12

Since ompR #73 and #64 aligned to ompR, we're cutting it out with XP to put the whole promoter-RBS-ompR-term part into a vector with either a CDF or SC101 origin. However, these digest products were inexplicable. One had a bright 4kb band and a faint 3kb band. The other had a bright 3kb band and a very faint kb band. Although the latter is similar to what is expected, the insert was extremely faint. Digest was repeated.

Image:8-12_gel_2_MXHTA.jpg

Lane 1: 1 kb ladder

Lane 2: P115 uncut (~3750)

Lane 3: P115A cut XP (~1000)

Lane 4: P115B cut XP (~1000)

Lane 5: P48 uncut (3477)

Lane 6: P48 cut XbaI (3477)

PCR

8/12

Since none of the alternative strategy mtrB or P105 is working, we tried to PCR out the parts again with Phusion, as it is a more processive polymerase and our products are all >2kb.

Phusion protocol says to use Tms ≥ 60°C, so we tried both a high and low temp set for each Rx. The low temp was optimal temp from using Platinum Taq; the high temp was the highest that indicated some form of a band.

The high temperature set used as template previous iterations of the same PCR. The low temperature set used either miniprep DNA or a colony.

Rx mix: 10μL 5x HF buffer
1μL 10mM dNTPs
1.25μL each primer
0.5μL template 0.5μL Phusion polymerase 35.5 H2O

P1/3

Rx conditions: 30s @ 98°C → 5x[10s @ 98°C → 30s @ (45 or 54)°C → 1min @ 72°C] → 10x[10s @ 98°C → 30s @ (45 or 54)°C → 1min5s @ 72°C] → 7min @ 72°C → ∞ @ 4°C

45°C worked- band purified, while 54°C did not (giant smear).

mtrB not BB

Rx conditions: 2:30s @ 98°C → 5x[10s @ 98°C → 30s @ (46 or 52)°C → 35s @ 72°C] → 10x[10s @ 98°C → 30s @ (46 or 52)°C → 40s @ 72°C] → 7min @ 72°C → ∞ @ 4°C

46°C worked- band purified, while 52°C did not (giant smear).

P105

P105C used for low temperature template.

Rx conditions: 2:30s @ 98°C → 5x[10s @ 98°C → 30s @ (57.8 or 60)°C → 35s @ 72°C] → 10x[10s @ 98°C → 30s @ (57.8 or 60)°C → 40s @ 72°C] → 7min @ 72°C → ∞ @ 4°C

57.8°C yielded a close double band a little bigger than 2kb (which is where the expected product is), while 60°C did not work (giant smear). We're not sure what the doublet is, so we are sending P105 for sequencing.

oompa loompas are yummy

Making Thermoinducible cI System

Ligation of P104 and P63+P18 with cI RBS from TOPO

Digestion of cI RBS from TOPO and P18+P63 8/8

P18+ P63 not confirmed by sequencing yet.

' cI857 RBS TOPO ES 1 cI857 RBS TOPO ES 2 cI857 RBS TOPO ES 3 cI857 RBS TOPO ES 5 P18+ P63 ES 1 P18+ P63 ES 2 P18+ P63 ES 3 P18+ P63 ES 4 E1 P63 + RBS PP B E1 P104 + RBS PP A E1 P63c + RBS PP G
DNAFill inFill inFill inFill inFill inFill inFill inFill inFill inFill inFill in
100X BSA0.25 uL0.25 uL0.25 uL0.25 uL0.25 uL0.25 uL0.25 uL0.25 uL0.25 uL0.25 uL0.25 uL
10X Buffer 22.5 uL2.5 uL2.5 uL2.5 uL2.5 uL2.5 uL2.5 uL2.5 uL2.5 uL of Buffer 32.5 uL of Buffer 32.5 uL of Buffer 3
EcoRI1 uL1 uL1 uL1 uL1 uL1 uL1 uL1 uL1 uL XbaI1 uL XbaI1 uL XbaI
Enzyme 21 uL SpeI1 uL SpeI1 uL SpeI1 uL SpeI1 uL XbaI1 uL XbaI1 uL XbaI1 uL XbaI1 uL PstI1 uL PstI1 uL PstI
WaterFill inFill inFill inFill inFill inFill inFill inFill inFill inFill inFill in
Volume25 uL25 uL25 uL25 uL25 uL25 uL25 uL25 uL25 uL25 uL25 uL

Gel results 8/9

Image:8-9-08_digests_al.jpg 1% agarose gel visualized using EtBr/UV
Lane Sample
11 kB Ladder
2cI857 RBS TOPO ES 1
3cI857 RBS TOPO ES 2
4cI857 RBS TOPO ES 3
5cI857 RBS TOPO ES 5
6
71 kB Ladder
8P18+ P63 EX 1
9P18+ P63 EX 2
10P18+ P63 EX 3
11P18+ P63 EX 4
121 kB Ladder
13E1 P63 + RBS PP B
14E1 P104 + RBS PP A
15E1 P63c + RBS PP G

RBS TOPO 1,3, and 5, and P18+ P63 EX 1-4 were extracted and purified.

Update: At least P18+ P63 1-2 have been sequence confirmed.

Digest of P104 and P63 8/9

' P63 EX P104 EX
DNA5 uL15 uL
100X BSA0.25 uL0.25 uL
10X Buffer 2.5 uL of Buffer 22.5 uL of Buffer 2
EcoRI1 uL1 uL
Enzyme 21 uL XbaI1 uL XbaI
Water15.25 uL5.25 uL
Volume25 uL25 uL

Gel of Digestion 8/10

Image:8-10-08_p63and104_ls.jpg 1% agarose gel visualized using EtBr/UV
Lane Sample
11 kB Ladder
2P104 EX
3P63 EX

Extracted and purified 8/10.

Ligation 8/11

Ligated P18 + P63 clones 1 and 2, and P104, with RBS from TOPO.

Plate Results 8/12

Plate Marker # Colonies Description
E1 P104 Dephos onlyKan0 colonies
E1 P104 + RBS TOPO 1Kan2 0.25 mm colonies?
E1 P104 + RBS TOPO 3Kan2-3 0.25mm colonies?
E1 P104 + RBS TOPO 5Kan1-2 0.25mm colonies?
E1 P104 + RBS TOPO 1 6:2Kan1-2 1mm colonies
E1 P63 + P18 Dephos onlyAmp0 colonies
E1 P63 + P18 + RBS TOPO 1Amp1 1mm colony, 1 2mm colony
E1 P63 + P18 + RBS TOPO 3Amp11 1mm coloniesnot evenly distributed
E1 P63 + P18 + RBS TOPO 5Amp5-6 0.25mm colonies, 1 0.5mm colony
E1 P63 + P18 + RBS TOPO 3 6:2Amp0 colonies
E1 P63 + P18 2 Dephos OnlyAmp2 0.25mm colonies
E1 P63 + P18 2 + RBS TOPO 1Amp22 1mm coloniesNot evenly distributed.
E1 P63 + P18 2 + RBS TOPO 3Amp~23 1mm coloniesNot evenly distributed.
E1 P63 + P18 2 + RBS TOPO 5Amp16 1mm colonies
Kan (-) ctrlKan~50 colonies
Amp (-) ctrlAmp0 colonies
puc 19 (+) ctrlAmp0 colonies

Colony PCR of Transformations 8/12

Image:8-13-08_colonypcr1_al.jpg 1% agarose gel visualized using EtBr/UV
Lane Sample
11 kb ladder
2E1 P104 + RBS TOPO 1 6:2
3E1 P63 + P18 (1) + RBS TOPO 1 A
4E1 P63 + P18 (1) + RBS TOPO 1 B
5E1 P63 + P18 (1) + RBS TOPO 3 A
6E1 P63 + P18 (1) + RBS TOPO 3 B
7E1 P63 + P18 (1) + RBS TOPO 5 A
8E1 P63 + P18 (2) + RBS TOPO 1 A
9E1 P63 + P18 (2) + RBS TOPO 1 B
10E1 P63 + P18 (2) + RBS TOPO 1 C
11E1 P63 + P18 (2) + RBS TOPO 3 A
12E1 P63 + P18 (2) + RBS TOPO 3 B
Image:8-13-08_colonypcr2_al.jpg 1% agarose gel visualized using EtBr/UV
Lane Sample
1E1 P63 + P18 (2) + RBS TOPO 3 C
2E1 P63 + P18 (2) + RBS TOPO 3 D
3E1 P63 + P18 (2) + RBS TOPO 5 A
4E1 P63 + P18 (2) + RBS TOPO 5 B
5E1 P63 + P18 (2) + RBS TOPO 5 C
61 kb Ladder
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