User:Karmella Haynes/Notebook/BioBrick cloning/2013/02/23

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(02/24/13)<br>
Golden Gate Reactions
Golden Gate Reactions
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{| {{table}} border="1" cellspacing="3" <!-- Golden Gate Rxn. table -->
{| {{table}} border="1" cellspacing="3" <!-- Golden Gate Rxn. table -->
|- valign="top"
|- valign="top"
-
| Reagent || 1 || 2
+
| Reagent || 1 || 2 || Master mix (x3)
|-
|-
-
| gg2 pSB1A3 || 1.0 || 1.0  
+
| gg2 pSB1A3 || 1.0 || 1.0 || ---
|-
|-
-
| gg3 hPCD || 1.0 || ---  
+
| gg3 LOV || 1.0 || H2O || ---
|-
|-
-
| gg4 BL01 || 1.0 || ---  
+
| gg4 H2B+his || 1.0 || H2O || ---
|-
|-
-
| 10x PRO ligase buffer || 1.0 || 1.0
+
| 10x PRO ligase buffer || 1.0 || 1.0 || 3.0
|-
|-
-
| NEB T4 lgase || 0.25 || 0.25
+
| NEB T4 lgase || 0.25 || 0.25 || 0.75
|-
|-
-
| BsmBI || 0.5 || 0.5
+
| BsmBI || 0.5 || 0.5 || 1.5
|-
|-
-
| dH<sub>2</sub>O || 5.25 || 7.25
+
| dH<sub>2</sub>O || 5.25 || 5.25 || 15.75
|-
|-
| &nbsp; || 10.0 || 10.0
| &nbsp; || 10.0 || 10.0
|}
|}
 +
* Aliquot 7.0 mix to each tube; add DNA/ H2O
Thermal cycling
Thermal cycling

Revision as of 21:34, 23 February 2013

Karmella's BioBrick Cloning Main project page
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02/23/13

  • Type IIS Assembly, PCR: LOV-H2B+his


Type IIS Assembly, PCR: LOV-H2B+his

  1. H2B = 378 bp, ggH2B f/r (Template: H2B-GFP from Addgene); note: reverse primer adds a 6-his tag (18 bp)
  2. LOV = 429 bp, ggLOV f/r (Template: LOV from Addgene)
  3. pSB1A3 = ~2000, gg0001/0002 v2

PCR

  • 95°C, 3 min.
  • [95°C, 30 sec.; 57°C, 30 sec.; 72°C, 1 min.] x35
  • 72°C, 3 min.
  • 4°C, ∞
Reagent Volume Expected:
1. H2B = 396
2. LOV = 429
3, 4. pSB1A3 = ~2000 		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


Purification

  • Clean with Zymo DNA c&c; elute w/ 20 μL dH2O
  • Measure [DNA]
Sample OD 260 260/280 ng/μL
1. H2B+his 0.04 1.667 40.4
2. LOV 0.019 1.562 18.6
3. pSB1A3 0.069 1.754 69.4
4. pSB1A3 0.085 1.817 85.5


Dilutions: to final concentration of 20 fmol/μL (40 μL)

DNA length (bp) ng/μL Vol. (x) + dH2O
pSB1A3 2000 85.5 12.2 27.8
H2B+his 396 40.4 5.1 34.9
LOV 429 18.6 12.0 28.0


(02/24/13)

Golden Gate Reactions

  1. LOV + H2B+his + pSB1A3
  2. pSB1A3
Reagent 1 2 Master mix (x3)
gg2 pSB1A3 1.0 1.0 ---
gg3 LOV 1.0 H2O ---
gg4 H2B+his 1.0 H2O ---
10x PRO ligase buffer 1.0 1.0 3.0
NEB T4 lgase 0.25 0.25 0.75
BsmBI 0.5 0.5 1.5
dH2O 5.25 5.25 15.75
  10.0 10.0
  • Aliquot 7.0 mix to each tube; add DNA/ H2O

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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