IGEM:Harvard/2008/Lab Notebooks/DailyBook/Week11/Chemical and Light

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(mtrB PCR 9/18)
(96 well e-gel: Colony PCR and (mtrB+RBS) PCR 09/19)
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=96 well e-gel: Colony PCR and (mtrB+RBS) PCR 09/19=
=96 well e-gel: Colony PCR and (mtrB+RBS) PCR 09/19=
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[[Image:9-19_96_well_gel.jpg]]
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Lanes 1-12: DNA +
Lanes 1-12: DNA +

Revision as of 14:54, 19 September 2008

Contents

Retransformations 9/15

P126 and 127 retransformed into DH5α. Split 30μL and rest onto 2 diff CM plates.

9/16: Retransform went fine, although still fewer cells/DNA could have been used. Colonies picked for overnight cultures.

Ligations 9/15

  • Tried the Takara ligation kit: 1μL vector, 4μL insert, 5μL ligation mix
  • Transformed into DH5α

mtrB

P63 EX (8/14, dephos) with:

  • mtrB TOPO ES (8/15): 0 colonies (on KAN)
  • mtrB BB ES (8/14): 3 colonies (on KAN)

P97 SP (dephos) with:

  • mtrB BB XP (8/14): TMTC (on AMP)
  • mtrB TOPO XP (8/15): TMTC (on AMP)

It seems like there's something wrong with the P97, so if the colony PCRs indicate there's nothing in the plasmids, the tube'll be tossed.

Colony PCR

The 3 mtrB+63 colonies and 4 of each mtrB+97 colonies were picked for colony PCR:

Mix (split into 11): 247.5μL Platinum supermix, 5.5μL BBpfx, 5.5μL BBsfx, 16.5μL H2O

Rx: 5min denature, 35 cycles of (45s denaturation at 94°C, 30s annealing at 55°C, 2m40s at 72°C), 7min final extension, hold at 4°C

Image:9-16_mtrB_mxh.jpg

2KB ladder used. Clearly, none of the clones actually had a 2kb+ band, so mtrB was not incorporated.

Lac QPI

P3+51 EX (8/3, dephos) with P38 oligo mix (Amy's prep)

0 colonies (on KAN)

RE digests 9/17: mtrB TOPO, 101, 102, 116, 117

The following digests were performed using the Fermentas protocol for 25 min: mtrB TOPO ES; mtrB TOPO XP; P63 EX; P97 SP, P101, 102, 116, 117 ES

Aside from 116, 117, the bands were extracted using a clonewell gel. 116 and 117 were not visible.

P63, P97, and P101 were dephosphorylated according to the NEB instructions using Antarctic phosphatase, and the phosphatase was heat killed.

Ligations 9/18

The following ligations were performed (using 1:6 vector:insert ratio):

  • mtrB TOPO ES + P63 EX
  • mtrB TOPO XP + P97 SP
  • P101 ES + P102 ES

The DNA was mixed, heated to 65°C for 2 minutes, and then mixed with ligase.

  • With Takara kit, 6μL DNA mix was added to 6μL ligation mix
  • With NEB kit, 9μL DNA mix was added to 1.1μL T4 ligation buffer and 1μL T4 ligase

The mix was cooled to 16°C for 45min and then held at 4°C prior to transformation.

mtrB PCR 9/18

Tried PCR again with primers that incorporate RBS and Platinum Pfx.

w/ cells w/ TOPO plasmid
12x12x
Amp buffer560Amp buffer560
10mM dNTP1.51810mM dNTP1.518
50 mM MgSO411250 mM MgSO4112
Primer F0.759Primer F0.759
Primer R0.759Primer R0.759
Pfx0.759Pfx0.759
Water40.25483Water40480
DNA0.253
w/PCR enhancer
Amp buffer560Amp buffer560
10mM dNTP1.51810mM dNTP1.518
50 mM MgSO411250 mM MgSO4112
Primer F0.759Primer F0.759
Primer R0.759Primer R0.759
Pfx0.759Pfx0.759
PCRx560Water35420
Water35.25423PCRx560
DNA0.253


Rx: 2min @ 94°C → 35x[15s @ 94°C → 30s @ {55-61}°C → 2m30s @ 68°C] → 7m @ 68°C → ∞ @ 4°C

96 well e-gel: Colony PCR and (mtrB+RBS) PCR 09/19

Image:9-19_96_well_gel.jpg


Lanes 1-12: DNA +

Lanes 13-24: DNA -

Lanes 25-36: cells +

Lanes 37-48: cells -

Lanes 49-60: 1-12 (colony PCR)

Lanes 61-72: 13-24 (colony PCR)

Lanes 73-84: 25-36 (colony PCR)

Lanes 85-95: 37-47 (colony PCR)

Lane 96: positive control (colony PCR)

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