Difference between revisions of "Team:UCLA/Notebook/Spider Silk Genetics/14 August 2015"

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=08/14/2015=
 
=08/14/2015=
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==Miniprep samples from [https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/13_August_2015 yesterday]==
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Sequencing results:
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M1-9(T7)
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* Colony 1,2 and 4 have the correct sequence
  
 
==PCR amplify M1-12==
 
==PCR amplify M1-12==
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| 0.5 uL
 
| 0.5 uL
 
|}
 
|}
 +
 +
{| class="wikitable"
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| 98C
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| 30 sec
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|-
 +
| 98C
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| 10 sec
 +
|-
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| 66C
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| 20 sec
 +
|-
 +
|
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| x23
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|-
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| 72C
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| 45 sec
 +
|-
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| 72C
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| 2 min
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|-
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| 12C
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| Hold
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|}
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 +
Ran samples on 1% gel
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[[File:2015-08-14 15hr 23min.jpg| 600px]]
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Sample 1 with had no band
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Sample 2 had a very bright band close to the expected size
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 +
Maybe it was the ICA elution that was no longer in good condition?
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 +
Gel purified the band from sample 2
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* 39.69 ng/uL; 260/280: 2.47
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==Double Digestion of M1-12 PCR product with EcoRI and PstI==
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{| "class="wikitable"
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| EcoRI
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| 1 uL
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|-
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| Pst I
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| 1 uL
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|-
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| Template
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| 11 uL
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|-
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| Buffer 2.1
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| 5 uL
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|-
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| ddH2)
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| 32 uL
 +
|}
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 +
37C for 1.5 hr, 65C for 20 min

Latest revision as of 22:39, 15 August 2015

iGEM UCLA




08/14/2015

Miniprep samples from yesterday

Sequencing results:

M1-9(T7)

  • Colony 1,2 and 4 have the correct sequence

PCR amplify M1-12

Since previous PCRs using ICA elution as template did not work very well, first M1-12 PCR product was also tested as a template

ICA elution PCR product
Q5 buff 10 uL 10 uL
dNTPs 1 uL 1 uL
F-03 2.5 uL 2.5 uL
G-03 2.5 uL 2.5 uL
ddH2O 32.5 uL 33.3 uL
Template 1 uL 0.2 uL
Q5 Polymerase 0.5 uL 0.5 uL
98C 30 sec
98C 10 sec
66C 20 sec
x23
72C 45 sec
72C 2 min
12C Hold

Ran samples on 1% gel

2015-08-14 15hr 23min.jpg

Sample 1 with had no band Sample 2 had a very bright band close to the expected size

Maybe it was the ICA elution that was no longer in good condition?

Gel purified the band from sample 2

  • 39.69 ng/uL; 260/280: 2.47

Double Digestion of M1-12 PCR product with EcoRI and PstI

EcoRI 1 uL
Pst I 1 uL
Template 11 uL
Buffer 2.1 5 uL
ddH2) 32 uL

37C for 1.5 hr, 65C for 20 min