User:Karmella Haynes/Notebook/BioBrick cloning/2015/04/23: Difference between revisions

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| Sample || OD260 || 260/280 || ng/μL
| Sample || OD260 || 260/280 || ng/μL
|-
|-
| 1. 5'p Gal4DB-mCh PCR || 0.139 || 1.825 || 138.6
| 1. AubR/MRV || 0.258 || 1.906 || 257.7
|-
|-
| 2. 5'p ATF2 PCR || 0.191 || 1.828 || 190.8
| 2. BjaR/MRV || 0.177 || 1.904 || 177.3
|-
|-
| 3. 5'p MV10 PCR || 0.066 || 1.716 || 65.9
| 3. RpaR/MRV || 0.246 || 1.896 || 246.2
|}
|}


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** Inserts are gBlocks = ~2ng/μL
** Inserts are gBlocks = ~2ng/μL
** For an insert = ~200 and vector = ~4000, need 5 ng insert for 2:1 insert:vector(50 ng) ratio
** For an insert = ~200 and vector = ~4000, need 5 ng insert for 2:1 insert:vector(50 ng) ratio
 
# pAub/AubR/MRV: pAub(E/Xdp)/153 + AubR/MRV(BbsI)/4087
# pBja/BjaR/MRV: pBja(E/Xdp)/122 + BjaR/MRV(BbsI)/3977
# pRpa/RpaR/MRV: pRpa(E/Xdp)/136 + RpaR/MRV(BbsI)/3980
* Note: BraR cloning was not successful, omit from this round of assembly


{| {{table}} cellspacing="3" <!-- Lig/Dig rxn table -->
{| {{table}} cellspacing="3" <!-- Lig/Dig rxn table -->
| &nbsp;            || 1    || 2    ||
| &nbsp;            || 1    || 2    || 3   
|-
|-
| Insert DNA        || 5.0  || --- ||
| Insert DNA        || 5.0  || 5.0 || 5.0
|-
|-
| Vector DNA        || ### || ### ||
| Vector DNA        || 0.2 || 0.3 || 0.2
|-
|-
| 2x lgn buf (Roche) || 7.5  || ### ||
| 10x buf (Roche) || 7.5  || 7.5 || 7.5
|-
|-
| T4 ligase (NEB)    || 1.0  || 1.0  ||
| T4 ligase (NEB)    || 1.0  || 1.0  || 1.0
|-
|-
| dH<sub>2</sub>O    || ###  || ###  ||
| dH<sub>2</sub>O    || ###  || ###  ||

Revision as of 17:15, 23 April 2015

Karmella's BioBrick Cloning <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
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04/23/15

  • Ryan - Receiver cloning stage 2


Ryan - Receiver plasmids, assembly

  • Stage 1 - insert Regulator ORFS
    • Phusion PCR-amplify ORFs (add 5'-E, 3'-X cut sites)
    • Insert ORF(E/X) into Vector (E/X). see Vector plasmid map
  • Stage 2 - insert Promoters
    • Cut & dephos promoters with E/X
    • Insert promoters(E/X) into Vector/Regulator (BbsI) via Lig/Dig cycling


Assemblies

  1. pAub/AubR/MRV: pAub(E/Xdp)/153 + AubR/MRV(BbsI)/4087
  2. pBja/BjaR/MRV: pBja(E/Xdp)/122 + BjaR/MRV(BbsI)/3977
  3. pBra/BraR/MRV: pBra(E/Xdp)/214 + BraR/MRV(BbsI)/3977
  4. pRpa/RpaR/MRV: pRpa(E/Xdp)/136 + RpaR/MRV(BbsI)/3980


  • Measure conc.'s
Sample OD260 260/280 ng/μL
1. AubR/MRV 0.258 1.906 257.7
2. BjaR/MRV 0.177 1.904 177.3
3. RpaR/MRV 0.246 1.896 246.2


  • Digests (Fermentas FD)
    • E/X
    • Inserts are gBlocks = 2.0 ng/μL
Reagent Volume
DNA (20 ng) 10.0 μL
10x buffer 2.0
EcoRI 1.0
XbaI 1.0
dH2O 6.0
  20.0 μL --> 37°C/ ~15 min.


  • Dephosphorylation (Roche)
    • Assuming that shrimp alkaline phosphatase works in FastDigest buffer conditions Link
Reagent Volume
DNA (digest) 20.0 μL (20 ng)
phosphatase 1.0
dH2O ---
  20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = ~1 ng/μL


  • Ligation/Digestion Reactions
    • Inserts are gBlocks = ~2ng/μL
    • For an insert = ~200 and vector = ~4000, need 5 ng insert for 2:1 insert:vector(50 ng) ratio
  1. pAub/AubR/MRV: pAub(E/Xdp)/153 + AubR/MRV(BbsI)/4087
  2. pBja/BjaR/MRV: pBja(E/Xdp)/122 + BjaR/MRV(BbsI)/3977
  3. pRpa/RpaR/MRV: pRpa(E/Xdp)/136 + RpaR/MRV(BbsI)/3980
  • Note: BraR cloning was not successful, omit from this round of assembly
  1 2 3
Insert DNA 5.0 5.0 5.0
Vector DNA 0.2 0.3 0.2
10x buf (Roche) 7.5 7.5 7.5
T4 ligase (NEB) 1.0 1.0 1.0
dH2O ### ###
  15.0 μL # μL

Thermal cycler: Bio-Rad C1000



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