User:Karmella Haynes/Notebook/BioBrick cloning/2013/02/23: Difference between revisions

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# LOV = 429 bp, ggLOV f/r (Template: LOV from Addgene)
# LOV = 429 bp, ggLOV f/r (Template: LOV from Addgene)
# pSB1A3 = ~2000, gg0001/0002 v2
# 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, ∞


{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
Line 36: Line 42:
| &nbsp; || 50.0 μL
| &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, ∞




Line 54: Line 54:
| bgcolor=#cfcfcf | ng/μL  
| bgcolor=#cfcfcf | ng/μL  
|-
|-
| 1. pSB1A3 || 0.126 || 1.888 || 77.357
| 1. H2B+his || 0.126 || 1.888 || 77.357
|-
| 2. LOV || 0.069 || 2.192 || 17.025
|-
|-
| 2. hPCD || 0.069 || 2.192 || 17.025
| 3. pSB1A3 || 0.121 || 1.898 || 66.878
|-
|-
| 3. BL01 || 0.121 || 1.898 || 66.878
| 4. pSB1A3 || 0.121 || 1.898 || 66.878
|}
|}


Line 73: Line 75:
| pSB1A3 || 2000 || 77.4 || 6.7 || 13.3
| pSB1A3 || 2000 || 77.4 || 6.7 || 13.3
|-
|-
| hPCD || 186 || 17.0 || 2.8 || 17.2
| H2B+his || 186 || 17.0 || 2.8 || 17.2
|-
|-
| BL01 || 2520 || 66.9 || 9.8 || 10.2
| LOV || 2520 || 66.9 || 9.8 || 10.2
|}
|}


Revision as of 16:48, 23 February 2013

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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 0.126 1.888 77.357
2. LOV 0.069 2.192 17.025
3. pSB1A3 0.121 1.898 66.878
4. pSB1A3 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
H2B+his 186 17.0 2.8 17.2
LOV 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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