User:Karmella Haynes/Notebook/BioBrick cloning/2013/01/09

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<!-- Precede finished items with a checkmark &#x2713; -->
<!-- Precede finished items with a checkmark &#x2713; -->
* Golden Gate assembly: hPCD + BL01 + pSB1A3
* Golden Gate assembly: hPCD + BL01 + pSB1A3
 +
* Violacein: plates from Tabor; incubate o/n
Line 16: Line 17:
* Use gg2 (pSB1A3), gg3, gg4; not gg1 (V0120) since it has BsmBI sites
* Use gg2 (pSB1A3), gg3, gg4; not gg1 (V0120) since it has BsmBI sites
-
Dilutions
+
* Dilutions
-
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
+
{| {{table}} border="1" cellspacing="3" <!-- Dilution table -->
|- valign="top"
|- valign="top"
| bgcolor=#cfcfcf | DNA
| bgcolor=#cfcfcf | DNA
-
| bgcolor=#cfcfcf | Conc.
+
| bgcolor=#cfcfcf | length (bp)
 +
| bgcolor=#cfcfcf | ng/μL
| bgcolor=#cfcfcf | Vol.
| bgcolor=#cfcfcf | Vol.
| bgcolor=#cfcfcf | + dH<sub>2</sub>O
| bgcolor=#cfcfcf | + dH<sub>2</sub>O
|-
|-
-
| gg2 pSB1A3 || 67.7 || ||  
+
| gg2 pSB1A3 || 2000 || 67.7 || 7.7 || 12.3
|-
|-
-
| gg3 hPCD || 22.5 || ||
+
| gg3 hPCD || 186 || 22.5 || 2.1 || 17.8
|-
|-
-
| gg4 BL01 || 55.9 || ||
+
| gg4 BL01 || 2520 || 55.9 || 11.7 || 8.3
|}
|}
 +
 +
* Golden Gate Reactions
 +
# hPCD + BL01 + pSB1A3
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# pSB1A3
 +
 +
{| {{table}} border="1" cellspacing="3" <!-- Golden Gate Rxn. table -->
 +
|- valign="top"
 +
| Reagent || 1 || 2
 +
|-
 +
| gg2 pSB1A3 || 1.0 || 1.0
 +
|-
 +
| gg3 hPCD || 1.0 || ---
 +
|-
 +
| gg4 BL01 || 1.0 || ---
 +
|-
 +
| 10x ligase buffer || 1.0 || 1.0
 +
|-
 +
| NEB T4 lgase || 0.25 || 0.25
 +
|-
 +
| BsmBI || 0.5 || 0.5
 +
|-
 +
| dH<sub>2</sub>O || 5.25 || 7.25
 +
|-
 +
| &nbsp; || 10.0 || 10.0
 +
|}
 +
 +
Thermal cycler
 +
* [45°C, 2 min.; 16°C, 5 min.] x25
 +
* 60°C, 20 min.
 +
* 80°C, 20 min.

Current revision

Karmella's BioBrick Cloning Main project page
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01/09/13

  • Golden Gate assembly: hPCD + BL01 + pSB1A3
  • Violacein: plates from Tabor; incubate o/n


Golden Gate Assembly

  • Use purified, measured PCR products from 1/05/13
  • Use gg2 (pSB1A3), gg3, gg4; not gg1 (V0120) since it has BsmBI sites
  • Dilutions
DNA length (bp) ng/μL Vol. + dH2O
gg2 pSB1A3 2000 67.7 7.7 12.3
gg3 hPCD 186 22.5 2.1 17.8
gg4 BL01 2520 55.9 11.7 8.3


  • Golden Gate Reactions
  1. hPCD + BL01 + pSB1A3
  2. pSB1A3
Reagent 1 2
gg2 pSB1A3 1.0 1.0
gg3 hPCD 1.0 ---
gg4 BL01 1.0 ---
10x ligase buffer 1.0 1.0
NEB T4 lgase 0.25 0.25
BsmBI 0.5 0.5
dH2O 5.25 7.25
  10.0 10.0

Thermal cycler

  • [45°C, 2 min.; 16°C, 5 min.] x25
  • 60°C, 20 min.
  • 80°C, 20 min.



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