Haynes Lab:Notebook/TB Biosensor/2012/06/08: Difference between revisions

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Line 15: Line 15:
| <u>Lane</u> || <u>Part Number</u> || <u>Gene(s)</u>  
| <u>Lane</u> || <u>Part Number</u> || <u>Gene(s)</u>  
|-
|-
|-1 || K118014 || RBS + crtE
| 1 || K118014 || RBS + crtE
|-
|-
|-2 || K274100 || RBS + crtE + RBS + crtI + RBS + crtB
| 2 || K274100 || RBS + crtE + RBS + crtI + RBS + crtB
|-
|-
|-3 || K274200 || RBS + crtE + RBS + crtB + RBS + crtI +RBS + crtY
| 3 || K274200 || RBS + crtE + RBS + crtB + RBS + crtI +RBS + crtY
|-
|-
|-4 || K118002 || crtB
| 4 || K118002 || crtB
|-
|-
|-5 || K118006 || RBS + crtB
| 5 || K118006 || RBS + crtB
|-
|-
|-6 || I742161 || RBS + crtI + RBS + crtB
| 6 || I742161 || RBS + crtI + RBS + crtB
|-
|-
|-7 || K118003 || crtI
| 7 || K118003 || crtI
|-
|-
|-8 || K118005 || RBS + crtI
| 8 || K118005 || RBS + crtI
|}
|}


Line 35: Line 35:
{| class="wikitable" border="0" cellspacing="3" <!-- PCR rxn. table -->
{| class="wikitable" border="0" cellspacing="3" <!-- PCR rxn. table -->
|-valign="top"
|-valign="top"
| <u>Gene</u> || <u>Plasmid</u> || <u>Primer F</u> || <u>Primer R</u>
| <u>Lane</u> || <u>Gene</u> || <u>Plasmid</u> || <u>Primer F</u> || <u>Primer R</u>
|-
|-
|crtE || 1 || P001 || P002
| 1 || crtE || 1 || P001 || P002
|-
|-
|crtE || 2 || P001 || P002
| 2 || crtE || 2 || P001 || P002
|-
|-
|crtE || 3 || P001 || P002
| 3 || crtE || 3 || P001 || P002
|-
|-
|crtB || 2 || P003 || P004
| 4 || crtB || 2 || P003 || P004
|-
|-
|crtB || 3 || P003 || P004
| 5 || crtB || 3 || P003 || P004
|-
|-
|crtB || 4 || P003 || P004
| 6 || crtB || 4 || P003 || P004
|-
|-
|crtB || 5 || P003 || P004
| 7 || crtB || 5 || P003 || P004
|-
|-
|crtI || 2 || P005 || P006
| 8 || crtI || 2 || P005 || P006
|-
|-
|crtI || 3 || P005 || P006
| 9 || crtI || 3 || P005 || P006
|-
|-
|crtI || 7 || P005 || P006
| 10 || crtI || 7 || P005 || P006
|-
|-
|crtI || 8 || P005 || P006
| 11 || crtI || 8 || P005 || P006
|}
|}


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|}
|}


--> Wang Lab PCR machine, block A
* 95°C/ 3 min.
* [95°C/ 30 sec; 50°C/ 30 sec; 72 °C/ 1 min.] x30
* 72 °C/ 3 min.
* 4 °C/ ∞


> Qiagen PCR cleanup kit; elute w/ 50ul EB<sub>2</sub>O<br>


<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->

Revision as of 17:11, 8 June 2012

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Entry title

PCR'd the plasmids cut on 12.05.29 to PCR out the individual genes with the correct overhangs (Silver Standard RFC[23] compatible with no frame shift).

Plasmids from 12.05.29:

Lane Part Number Gene(s)
1 K118014 RBS + crtE
2 K274100 RBS + crtE + RBS + crtI + RBS + crtB
3 K274200 RBS + crtE + RBS + crtB + RBS + crtI +RBS + crtY
4 K118002 crtB
5 K118006 RBS + crtB
6 I742161 RBS + crtI + RBS + crtB
7 K118003 crtI
8 K118005 RBS + crtI


Lane Gene Plasmid Primer F Primer R
1 crtE 1 P001 P002
2 crtE 2 P001 P002
3 crtE 3 P001 P002
4 crtB 2 P003 P004
5 crtB 3 P003 P004
6 crtB 4 P003 P004
7 crtB 5 P003 P004
8 crtI 2 P005 P006
9 crtI 3 P005 P006
10 crtI 7 P005 P006
11 crtI 8 P005 P006


PCR reaction

Reagent Volume
DNA (plasmid) 1.0
10 μM primers 1.0
2x GoTaq 25.0
dH2O 23.0

--> Wang Lab PCR machine, block A

  • 95°C/ 3 min.
  • [95°C/ 30 sec; 50°C/ 30 sec; 72 °C/ 1 min.] x30
  • 72 °C/ 3 min.
  • 4 °C/ ∞

> Qiagen PCR cleanup kit; elute w/ 50ul EB2O


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