Difference between revisions of "IGEM:Harvard/2008/Lab Notebooks/DailyBook/Week10/Chemical and Light"

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(Getting mtrB onto thermo and lac inducible systems)
(Getting Thermoinducible and Lac Inducible GFP into pSC101)
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=Getting Thermoinducible and Lac Inducible GFP into pSC101=
 
=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===
 +
 +
{| {{table}}
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!rowspan=11|[[Image:8-27-08_amy_digests.jpg]]
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!colspan=2 align="center" style="background:#f0f0f0;"|'''1% agarose'''
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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 + DNA Ladder
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|-
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| 2||P124 1:2.5 A
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|-
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| 3||P124 1:2.5 B
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|-
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| 4||P125 1:2.5 Dephos A
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|-
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| 5||P125 1:2.5 D
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|-
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| 6||mtrB TOPO 0.5 uL B
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|-
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| 7||mtrB TOPO 2 uL B
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|-
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| 8||mtrB TOPO 4-1
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|-
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| 9||mtrB TOPO 4-10
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|}
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 +
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===
  
 
=Getting mtrB onto thermo and lac inducible systems=
 
=Getting mtrB onto thermo and lac inducible systems=

Revision as of 22:38, 30 August 2008

Transformations in Shewie

About 250 ng of DNA added each time.

First Set 8/22

Plate Colonies Size Color
S1 P102 (P108+45) Kan 5 colonies 3mm to 5 mm Pink
S1 P121+P38 (P124) 1:2.5 Kan A 1 colony 5mm to 7.5mm Pink
S1 P28 (ColE1) Kan 0 colonies
S1 P121+P38(P124) 1:2.5 Kan B 0 colonies
S1 P121+P39 1:2.5 Dephos Kan A 0 colonies
S1 Topo vector (puc) 2ul Kan X-gal >50 colonies 1mm White
S1 Topo vector (puc) 4ul Kan X-gal >50 colonies 1mm White
S1 P121+P39 1:2.5 Kan D 0 colonies
S1 (-) ctrl (just cells) Kan 0 colonies
S1 (+) ctrl (E1 p59b) Kan 2 colonies 5mm 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 A Kan 14 2-3mm pink colonies, ~100 0.5mm white colonies
S1 P124 1:2.5 B Kan 2 pink 2mm colonies, >100 0.5mm white colonies
S1 P125 1:2.5 Dephos A Kan 2 pink 2mm colonies, >100 0.5mm white colonies
S1 P125 1:2.5 D Kan 8 2mm pink colonies, ~100 0.5mm white colonies
S1 P28 Kan ~8 1-2mm pink colonies, ~50 0.5mm white colonies
S1 TOPO 2uL Kan Lawn of (>200) 0.5mm white colonies
S1 TOPO 4 uL (w/ Xgal) Kan Lawn of (>200) 0.5mm white colonies
S1 P59b (+) ctrl Kan ~50 2mm pink colonies, ~50 0.5mm white colonies
S1 (-) ctrl Kan 50-100 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

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

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.