User:Karmella Haynes/Notebook/BioBrick cloning/2015/05/15: Difference between revisions

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| rowspan="7" | <!-- [[Image:GelImage.jpg|270px|Hover name]]<br>30 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] -->
| rowspan="7" | <!-- [[Image:GelImage.jpg|270px|Hover name]]<br>30 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] -->
|-
|-
| DNA (plasmid) || up to 25 μL
| DNA (plasmid) || 15.0
|-
|-
| 10x buffer || 3.0
| 10x buffer (FD) || 3.0
|-
|-
| enzyme 1 || 1.0
| enzyme 1 || 1.0
|-
|-
| enzyme 2 || 1.0
| dH<sub>2</sub>O || 11.0
|-
| dH<sub>2</sub>O || ---
|-
|-
| &nbsp; || 30 μL --> 37°C/ ~30 min.
| &nbsp; || 30 μL --> 37°C/ ~30 min.

Revision as of 11:34, 15 May 2015

Karmella's BioBrick Cloning <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
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05/15/15

  • Ryan - assembly (traditional) for Receiver plasmids
  • Ben - assembly for luc replacement
  • Rene - PCR library transformation (quick); use remainder of reactions 3 & 4
  • Cas-tone - minipreps & digests


Assemblies

  • For Ryan and Ben
  1. pAub-AubR_MRV: pAub-PCR(E/S dp)/153 + AubR/MRV(BbsI)/4087
  2. pBja-BjaR_MRV: pBja-PCR(E/S dp) + BjaR/MRV(BbsI)/3977
  3. pBra-BraR_MRV: pBra-PCR(E/S dp) + BraR/MRV(BbsI)/3977
  4. pRpa-RpaR_MRV: pRpa-PCR(E/S dp) + RpaR/MRV(BbsI)/3980
  5. DBN007_pSB1A3: DBN006(BsaI)/1972 + DBN001(BsaI)/1267/1069(2336)


  • Digests (Fermentas FD)
  1. AubR/MRV(BbsI)/4087
  2. BjaR/MRV(BbsI)/3977
  3. BraR/MRV(BbsI)/3977
  4. RpaR/MRV(BbsI)/3980
  5. DBN006(BsaI)/1972
  6. DBN001(BsaI)/1267/1069(2336) (two bands)


Reagent Volume
DNA (plasmid) 15.0
10x buffer (FD) 3.0
enzyme 1 1.0
dH2O 11.0
  30 μL --> 37°C/ ~30 min.


  • Measure conc.'s
Sample OD260 260/280 ng/μL
1. Digested part (a/b) --- --- ---
2. Digested part (c/d) --- --- ---


  • Dephosphorylation (Roche)
Reagent Volume
DNA (clean digest) up to 17 μL (500 ng)
10x buffer d.p. 2.0
phosphatase 1.0
dH2O ---
  20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL


  • Ligations
Ligation Plate results (lig : neg crtl) mm/dd/yy
1. insert(a/b)/size, ## ng + vector(c/d)/size, ## ng new BioBrick #:1 (Pick #)
2. vector(c/d)/ ## ng  
  1 2
Insert DNA ### ---
Vector DNA ### ###
2x lgn buf (Roche) ### ###
T4 ligase (NEB) 1.0 1.0
dH2O ### ###
  # μL # μL

Oligo annealing

  1. New BB 1
  2. New BB 2
DNA (oligos, 100 μM) up to 18 μL (3 μL ea.)
10x annealing buffer 2.0
dH2O ---
  20 μL --> 100°C (water bath)/ 5 min.; Cool to R.T. overnight



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