Difference between revisions of "User:Karmella Haynes/Notebook/BioBrick cloning/2013/08/29"

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(08/29/13)
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----
 
----
'''Golden gate'''
+
'''Golden Gate'''
* Repeat procedure from ###
+
* Try to increase robustness by using more T4 ligase
 
+
# hPCD + BL01 + pSB1A3
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
+
# pSB1A3 alone (neg ctrl)
|- valign="top"
 
| bgcolor=#cfcfcf | Reagent
 
| bgcolor=#cfcfcf | Volume
 
| rowspan="7" | <u>Expected:</u><br>1. pSB1A3 = ~2000<br>2. hPCD = 186<br>3. BL01 = 2520
 
| 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
 
|-
 
| primer 1 || 1.0
 
|-
 
| primer 2 || 1.0
 
|-
 
| 2x GoTaq || 25.0
 
|-
 
| dH<sub>2</sub>O || 22.8
 
|-
 
| &nbsp; || 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 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
 
|}
 
  
 +
* GG-PCR fragments generated on 1/10/13
 +
** Diluted to 20 fmol/μL
  
 
Golden Gate Reactions
 
Golden Gate Reactions
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|}
 
|}
  
Thermal cycling
+
GG Thermal cycling
 
* [45°C, 2 min.; 16°C, 5 min.] x25
 
* [45°C, 2 min.; 16°C, 5 min.] x25
 
* 60°C, 20 min.
 
* 60°C, 20 min.

Revision as of 12:27, 29 August 2013

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08/29/13

  • Golden Gate Assembly optimization - increase ligase amount; redo hPCD assembly



Golden Gate

  • Try to increase robustness by using more T4 ligase
  1. hPCD + BL01 + pSB1A3
  2. pSB1A3 alone (neg ctrl)
  • GG-PCR fragments generated on 1/10/13
    • Diluted to 20 fmol/μL

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

GG Thermal cycling

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