IGEM:Harvard/2008/Lab Notebooks/DailyBook/Week10/Chemical and Light

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(Getting Thermoinducible and Lac Inducible GFP into pSC101)
(Single-cut Digest of P124-5 8/27)
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===Single-cut Digest of P124-5 8/27===
===Single-cut Digest of P124-5 8/27===
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{| {{table}}
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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 + DNA Ladder
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|-
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| 2||P124 1:2.5 A with XbaI
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|-
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| 3||P124 1:2.5 B with XbaI
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|-
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| 4||P125 1:2.5 Dephos A with XbaI
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|-
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| 5||P125 1:2.5 D with XbaI
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|-
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| 7||P124 1:2.5 A with PstI
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|-
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| 8||P124 1:2.5 B  with PstI
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|-
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| 9||P125 1:2.5 Dephos A  with PstI
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|-
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| 10||P125 1:2.5 D  with PstI
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|-
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| 11||1 kb + DNA Ladder
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|}
=Getting mtrB onto thermo and lac inducible systems=
=Getting mtrB onto thermo and lac inducible systems=

Revision as of 00:41, 31 August 2008

Contents

Transformations in Shewie

About 250 ng of DNA added each time.

First Set 8/22

Plate Colonies Size Color
S1 P102 (P108+45) Kan5 colonies3mm to 5 mmPink
S1 P121+P38 (P124) 1:2.5 Kan A1 colony5mm to 7.5mmPink
S1 P28 (ColE1) Kan0 colonies
S1 P121+P38(P124) 1:2.5 Kan B0 colonies
S1 P121+P39 1:2.5 Dephos Kan A0 colonies
S1 Topo vector (puc) 2ul Kan X-gal>50 colonies1mmWhite
S1 Topo vector (puc) 4ul Kan X-gal>50 colonies1mmWhite
S1 P121+P39 1:2.5 Kan D0 colonies
S1 (-) ctrl (just cells) Kan0 colonies
S1 (+) ctrl (E1 p59b) Kan2 colonies5mm Pink

Poor transformation efficiency in the positive control suggests that there may be a problem with our cells. All samples, except for P102, were retransformed the following week with fresh cell cultures.

Second Set (with new cells) 8/27

Plate Marker Description
S1 P124 1:2.5 AKan14 2-3mm pink colonies, ~100 0.5mm white colonies
S1 P124 1:2.5 BKan2 pink 2mm colonies, >100 0.5mm white colonies
S1 P125 1:2.5 Dephos AKan2 pink 2mm colonies, >100 0.5mm white colonies
S1 P125 1:2.5 DKan8 2mm pink colonies, ~100 0.5mm white colonies
S1 P28Kan~8 1-2mm pink colonies, ~50 0.5mm white colonies
S1 TOPO 2uLKanLawn of (>200) 0.5mm white colonies
S1 TOPO 4 uL (w/ Xgal)KanLawn of (>200) 0.5mm white colonies
S1 P59b (+) ctrlKan~50 2mm pink colonies, ~50 0.5mm white colonies
S1 (-) ctrlKan50-100 0.5mm white colonies

Getting Thermoinducible and Lac Inducible GFP into pSC101

Digests of Inducible Systems and pSC101 8/27

P102 needs to be retransformed so it was not included in this set.

Gel of Digest 8/27

Image:8-27-08_amy_digests.jpg 1% agarose
Lane Sample
11 kb + DNA Ladder
2P124 1:2.5 A
3P124 1:2.5 B
4P125 1:2.5 Dephos A
5P125 1:2.5 D
6mtrB TOPO 0.5 uL B
7mtrB TOPO 2 uL B
8mtrB TOPO 4-1
9mtrB TOPO 4-10

mtrB TOPO bands were cut (2.2 kB) but P124-5 did not cut properly, suggesting that there was an extra cut site that was cutting the insert into two pieces.

Single-cut Digest of P124-5 8/27

Lane Sample
11 kb + DNA Ladder
2P124 1:2.5 A with XbaI
3P124 1:2.5 B with XbaI
4P125 1:2.5 Dephos A with XbaI
5P125 1:2.5 D with XbaI
7P124 1:2.5 A with PstI
8P124 1:2.5 B with PstI
9P125 1:2.5 Dephos A with PstI
10P125 1:2.5 D with PstI
111 kb + DNA Ladder

Getting mtrB onto thermo and lac inducible systems

Ligating mtrB TOPO with RBS

Grew up more mtrB TOPO for next week.

PCR of mtrB with RBS + mtrB primers

Scheduled for next week.

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