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

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'''Type IIS Assembly, PCR: LOV-H2B+his'''
'''Type IIS Assembly, PCR: LOV-H2B+his'''
# H2B = 378 bp, 1A3/H2B fwd + H2B/LOV rev (Template: H2B-GFP from Addgene)
# H2B = 378 bp, 1A3/H2B fwd + H2B/LOV rev (Template: H2B-GFP from Addgene)
# LOV = 429 bp, LOV fwd + LOV+his/1A3 rev (Template: LOV from Addgene); note: reverse primer adds a 6-his tag (18 bp)
# LOV = 429 bp, LOV fwd + LOV+his/1A3 rev (Template: LOV from Addgene); note: reverse primer adds a 6-his tag and TAA stop (21 bp)
# pSB1A3 = ~2000, gg0001 v2 + gg0002 v2
# pSB1A3 = ~2000, gg0001 v2 + gg0002 v2
* Be sure to use the H2B and LOV primers ordered on 2/4/2003. The names are redundant with a previous version.
* Be sure to use the H2B and LOV primers ordered on 2/4/2003. The names are redundant with a previous version.
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| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume  
| bgcolor=#cfcfcf | Volume  
| rowspan="7" | <u>Expected:</u><br>1. H2B = 378<br>2. LOV = 447<br>3, 4. pSB1A3 = ~2000
| rowspan="7" | <u>Expected:</u><br>1. H2B = 378<br>2. LOV = 450<br>3, 4. pSB1A3 = ~2000
| rowspan="7" | [[Image:KAH022313_gel1.jpg|200px|Hover name]]<br>10 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
| rowspan="7" | [[Image:KAH022313_gel1.jpg|200px|Hover name]]<br>10 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
|-
|-

Revision as of 12:02, 25 February 2013

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02/23/13

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


Type IIS Assembly, PCR: LOV-H2B+his

  1. H2B = 378 bp, 1A3/H2B fwd + H2B/LOV rev (Template: H2B-GFP from Addgene)
  2. LOV = 429 bp, LOV fwd + LOV+his/1A3 rev (Template: LOV from Addgene); note: reverse primer adds a 6-his tag and TAA stop (21 bp)
  3. pSB1A3 = ~2000, gg0001 v2 + gg0002 v2
  • Be sure to use the H2B and LOV primers ordered on 2/4/2003. The names are redundant with a previous version.

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 = 378
2. LOV = 450
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)

  • x = length in bp ÷ measured ng/μL * 0.013 * 40
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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