User:Karmella Haynes/Notebook/BioBrick cloning/2013/01/10

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'''Golden gate trial 1'''
'''Golden gate trial 1'''
* Add total 10.0 μL run to 50 μL DH5α-Turbo; incubate on ice 10 min.; plate on amp agar
* Add total 10.0 μL run to 50 μL DH5α-Turbo; incubate on ice 10 min.; plate on amp agar
 +
* Colonies: 7 on assembly plate, 9 on vector-only control. Pick 4 and make 2 mL liquid cultures.
Line 33: Line 34:
| bgcolor=#cfcfcf | Volume  
| bgcolor=#cfcfcf | Volume  
| rowspan="7" | <u>Expected:</u><br>1. pSB1A3 = ~2000<br>2. hPCD = 186<br>3. BL01 = 2520
| rowspan="7" | <u>Expected:</u><br>1. pSB1A3 = ~2000<br>2. hPCD = 186<br>3. BL01 = 2520
-
| rowspan="7" | <!-- [[Image:GelImage.jpg|400px|Hover name]]<br>15 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] -->
+
| rowspan="7" | [[Image:KAH011013_gel1.jpg|200px|Hover name]]<br>10 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
|-
|-
| DNA(plasmid) || 0.2 μL
| DNA(plasmid) || 0.2 μL
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PCR
PCR
* 95°C, 3 min.
* 95°C, 3 min.
-
* [95°C, 3 min.; 95°C, 3 min.; 95°C, 3 min.] x35
+
* [95°C, 30 sec.; 57°C, 30 sec.; 72°C, 1 min.] x35
* 72°C, 3 min.
* 72°C, 3 min.
 +
* 4°C, ∞
 +
 +
 +
Purification
 +
* Clean with Zymo DNA c&c; elute w/ 20 μL dH<sub>2</sub>O
 +
* Measure [DNA]
 +
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
 +
|- valign="top"
 +
| bgcolor=#cfcfcf | Sample
 +
| bgcolor=#cfcfcf | OD 260
 +
| bgcolor=#cfcfcf | 260/280
 +
| bgcolor=#cfcfcf | ng/μL
 +
|-
 +
| 1. pSB1A3 || 0.126 || 1.888 || 77.357
 +
|-
 +
| 2. hPCD || 0.069 || 2.192 || 17.025
 +
|-
 +
| 3. BL01 || 0.121 || 1.898 || 66.878
 +
|}
 +
 +
 +
Dilutions: to final concentration of 20 fmol/μL
 +
{| {{table}} border="1" cellspacing="3" <!-- Dilution table -->
 +
|- valign="top"
 +
| bgcolor=#cfcfcf | DNA
 +
| bgcolor=#cfcfcf | length (bp)
 +
| bgcolor=#cfcfcf | ng/μL
 +
| bgcolor=#cfcfcf | Vol.
 +
| bgcolor=#cfcfcf | + dH<sub>2</sub>O
 +
|-
 +
| pSB1A3 || 2000 || 77.4 || 6.7 || 13.3
 +
|-
 +
| hPCD || 186 || 17.0 || 2.8 || 17.2
 +
|-
 +
| BL01 || 2520 || 66.9 || 9.8 || 10.2
 +
|}
 +
 +
 +
Golden Gate Reactions
 +
# hPCD + BL01 + pSB1A3
 +
# 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 cycling
 +
* [45°C, 2 min.; 16°C, 5 min.] x25
 +
* 60°C, 20 min.
 +
* 80°C, 20 min.
* 4°C, ∞
* 4°C, ∞

Current revision

Karmella's BioBrick Cloning Main project page
Previous entry      Next entry

01/10/13

  • Violacein: successful growth; re-streak
  • Golden gate/ transformation: transform DH5α-Turbo with gg assemblies from 1/09/13
  • Golden gate trial 2: new PCR primers



Violacein

  • Plate 1: Kan plasmid
  • Plate 2: Amp plasmid (violacein exp.)



Golden gate trial 1

  • Add total 10.0 μL run to 50 μL DH5α-Turbo; incubate on ice 10 min.; plate on amp agar
  • Colonies: 7 on assembly plate, 9 on vector-only control. Pick 4 and make 2 mL liquid cultures.



Golden gate trial 2

  • Re-designed primers to include "handle" preceding the BsmBI sites and a spacer between BsmBI and a 4 bp overlap region
Reagent Volume Expected:
1. pSB1A3 = ~2000
2. hPCD = 186
3. BL01 = 2520
Hover name
10 μL/lane; 1% agarose; Ladder
DNA(plasmid) 0.2 μL
primer 1 1.0
primer 2 1.0
2x GoTaq 25.0
dH2O 22.8
  50.0 μL

PCR

  • 95°C, 3 min.
  • [95°C, 30 sec.; 57°C, 30 sec.; 72°C, 1 min.] x35
  • 72°C, 3 min.
  • 4°C, ∞


Purification

  • Clean with Zymo DNA c&c; elute w/ 20 μL dH2O
  • Measure [DNA]
Sample OD 260 260/280 ng/μL
1. pSB1A3 0.126 1.888 77.357
2. hPCD 0.069 2.192 17.025
3. BL01 0.121 1.898 66.878


Dilutions: to final concentration of 20 fmol/μL

DNA length (bp) ng/μL Vol. + dH2O
pSB1A3 2000 77.4 6.7 13.3
hPCD 186 17.0 2.8 17.2
BL01 2520 66.9 9.8 10.2


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 cycling

  • [45°C, 2 min.; 16°C, 5 min.] x25
  • 60°C, 20 min.
  • 80°C, 20 min.
  • 4°C, ∞



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