Exeter/24 July 2013

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(Created page with " == Minipreps using the Qiagen kit == We miniprepped: * CcaR * Promoter, RBS x 3 replicates * CcaS * B0034 * Magenta * OmpR promoter * OmpR * YF1 * Lambda inverta * F...")
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== Minipreps using the Qiagen kit  ==
== Minipreps using the Qiagen kit  ==
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== NanoDrop from miniprep ==
== NanoDrop from miniprep ==
 +
 +
{| class="wikitable"
 +
|-
 +
! Part !! Concentration (ng/ul)
 +
|-
 +
| CcaR ||324.6
 +
|-
 +
| CcaS || 264.4
 +
|-
 +
| Yellow || 64.3
 +
|-
 +
| Magenta || 102.7
 +
|-
 +
| RBS || 48.9
 +
|-
 +
| Promoter, RBS, #1 || 21.5
 +
|-
 +
| Promoter, RBS, #2 || 22.6
 +
|-
 +
| Promoter, RBS, #3 || 22.0
 +
|-
 +
| Fix L || 31.9
 +
|-
 +
| Fix J || 35.4
 +
|-
 +
| YF1 || 27.1
 +
|-
 +
| OmpF || 28.2
 +
|}
 +
 +
 +
No sure clean was needed.
 +
 +
 +
We then cut the genes out of the plasmid using Xbal and Pstl.  Prepare to run on a gel.
 +
 +
Each eppendorf contains:
 +
 +
* 12ul purite water
 +
 +
* 2ul 10 fast digest buffer w.Green
 +
 +
* 0.5ul Xbal
 +
 +
* 0.5ul Pstl
 +
 +
* 5ul DNA
 +
 +
== In gel ==
 +
 +
{| class="wikitable"
 +
|-
 +
! Lane !! Content
 +
|-
 +
| 1 || Ladder (1kb Gene Ruler)
 +
|-
 +
| 2 || Promoter, RBS, #1
 +
|-
 +
| 3 || B0034
 +
|-
 +
| 4 || CcaS
 +
|-
 +
| 5 || Fix L
 +
|-
 +
| 6 || Yellow
 +
|-
 +
| 7 || Promoter, RBS, #2
 +
|-
 +
| 8 || Fix J
 +
|-
 +
| 9 || OmpF
 +
|-
 +
| 10 || Promoter, RBS, #3
 +
|-
 +
| 11 || Magenta
 +
|-
 +
| 12 || CcaR
 +
|-
 +
| 13 || YF1
 +
|-
 +
| 14 || Ladder (1kb Gene Ruler)
 +
|}
 +
 +
 +
We also ran a seperate gel of:
 +
 +
* cph8 #3
 +
 +
* cph8 #4
 +
 +
* cph8 #5
 +
 +
* cph8 #6
 +
 +
* cph8 #7
 +
 +
Cut with Xbal and Pstl.
 +
 +
The lanes were run in the order above, flanked by a 1kb Gene ruler.

Revision as of 11:47, 25 July 2013

Contents

Minipreps using the Qiagen kit

We miniprepped:

  • CcaR
  • Promoter, RBS x 3 replicates
  • CcaS
  • B0034
  • Magenta
  • OmpR promoter
  • OmpR
  • YF1
  • Lambda inverta
  • Fix J
  • Yellow
  • Fix J promoter


Glycerol stocks

We made glycerol stocks of what is above, then stored them at -80oC

For future reference, to get glycerol stocks back as plates, take a loop and streak on gel. Has to be taken from frozen glycerol stocks, which are immediately returned to the freezer.


We then made a gel for digests using ethidium bromide.


NanoDrop from miniprep

Part Concentration (ng/ul)
CcaR 324.6
CcaS 264.4
Yellow 64.3
Magenta 102.7
RBS 48.9
Promoter, RBS, #1 21.5
Promoter, RBS, #2 22.6
Promoter, RBS, #3 22.0
Fix L 31.9
Fix J 35.4
YF1 27.1
OmpF 28.2


No sure clean was needed.


We then cut the genes out of the plasmid using Xbal and Pstl. Prepare to run on a gel.

Each eppendorf contains:

  • 12ul purite water
  • 2ul 10 fast digest buffer w.Green
  • 0.5ul Xbal
  • 0.5ul Pstl
  • 5ul DNA

In gel

Lane Content
1 Ladder (1kb Gene Ruler)
2 Promoter, RBS, #1
3 B0034
4 CcaS
5 Fix L
6 Yellow
7 Promoter, RBS, #2
8 Fix J
9 OmpF
10 Promoter, RBS, #3
11 Magenta
12 CcaR
13 YF1
14 Ladder (1kb Gene Ruler)


We also ran a seperate gel of:

  • cph8 #3
  • cph8 #4
  • cph8 #5
  • cph8 #6
  • cph8 #7

Cut with Xbal and Pstl.

The lanes were run in the order above, flanked by a 1kb Gene ruler.

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