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

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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Karmella's BioBrick Cloning</span>
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Karmella's BioBrick Cloning</span>
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
|style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}}
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<!-- Precede finished items with a checkmark &#x2713; -->
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* Ryan - Receiver cloning stage 2
* Ryan - Receiver cloning stage 2
* Rene - sequencing for g053-2, g054




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* Stage 2 - insert Promoters
* Stage 2 - insert Promoters
** '''Cut & dephos promoters with E/X'''
** '''Cut & dephos promoters with E/X'''
** Insert promoters(E/X) into Vector/Regulator (BbsI) via Lig/Dig cycling
** '''Insert promoters(E/X) into Vector/Regulator (BbsI) via Lig/Dig cycling'''




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| dH<sub>2</sub>O || ---
| dH<sub>2</sub>O || ---
|-
|-
| &nbsp; || 20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = ~1 ng/μL
| &nbsp; || 20 μL  
|}
|}
--> 37°C/ 10 min.; 75°C/ 2 min.; [final] = ~1 ng/μL






* Ligation/Digestion Reactions
* Digestion/Ligation Reactions (borrowed from Rene's gRNA/Cas9 assembly procedure)
** 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
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| Vector DNA        || 0.2  || 0.3  || 0.2
| Vector DNA        || 0.2  || 0.3  || 0.2
|-
|-
| 10x buf (Roche) || 7.5 || 7.5 || 7.5
| 10x FD buf         || 2.0 || 2.0 || 2.0
|-
|-
| T4 ligase (NEB)    || 1.0 || 1.0 || 1.0
| 10 mM DTT          || 1.0 || 1.0 || 1.0
|-
|-
| dH<sub>2</sub>O    || ###  || ###  ||
| 10 mM ATP          || 1.0 || 1.0 || 1.0
|-
|-
| &nbsp;            || 15.0 μL || # μL ||
| FD BbsI/BpiI      || 1.0  || 1.0  || 1.0
|-
| T4 ligase (Roche)  || 1.0  || 1.0  || 1.0
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| dH<sub>2</sub>O    || 8.8  || 8.7  || 8.8
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| &nbsp;            || 20.0 μL || 20.0 μL || 20.0 μL ||
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Thermal cycler: Bio-Rad C1000
Thermal cycler: Labnet - BbsI Dig/Lig
*  
* 6x[37°C, 5 min.; 23°C 5 min.]
* 4°C, ∞
 
 
----
'''Rene - sequencing'''
 
# g53-2, f (P139) - Confirmed
# g53-2, r (P140) - low Phred 20 score
# g54, f (P139) - Confirmed
# g54, r (P140) - low Phred 20 score
 
 




Latest revision as of 00:55, 27 September 2017

Karmella's BioBrick Cloning Main project page
Previous entry      Next entry

04/23/15

  • Ryan - Receiver cloning stage 2
  • Rene - sequencing for g053-2, g054


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


  • Digestion/Ligation Reactions (borrowed from Rene's gRNA/Cas9 assembly procedure)
    • 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 FD buf 2.0 2.0 2.0
10 mM DTT 1.0 1.0 1.0
10 mM ATP 1.0 1.0 1.0
FD BbsI/BpiI 1.0 1.0 1.0
T4 ligase (Roche) 1.0 1.0 1.0
dH2O 8.8 8.7 8.8
  20.0 μL 20.0 μL 20.0 μL

Thermal cycler: Labnet - BbsI Dig/Lig

  • 6x[37°C, 5 min.; 23°C 5 min.]
  • 4°C, ∞


Rene - sequencing

  1. g53-2, f (P139) - Confirmed
  2. g53-2, r (P140) - low Phred 20 score
  3. g54, f (P139) - Confirmed
  4. g54, r (P140) - low Phred 20 score




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