User:Karmella Haynes/Notebook/BioBrick cloning/2015/04/28: 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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| bgcolor=#cfcfcf | Volume  
| bgcolor=#cfcfcf | Volume  
| rowspan="7" | <u>Expected:</u><br>1-4. Bra/MRV = ~3200, 776<br>Empty MRV E/X = ~3200<br>5. pAub/AubR/MRV = 4240<br>6. pBja/BjaR/MRV = 4099<br>7. pRpa/RpaR/MRV = 4116<br>Empty vector = ~4000, 971
| rowspan="7" | <u>Expected:</u><br>1-4. Bra/MRV = ~3200, 776<br>Empty MRV E/X = ~3200<br>5. pAub/AubR/MRV = 4240<br>6. pBja/BjaR/MRV = 4099<br>7. pRpa/RpaR/MRV = 4116<br>Empty vector = ~4000, 971
| rowspan="7" | <!-- [[Image:GelImage.jpg|400px|Hover name]]<br>15 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] -->
| rowspan="7" | [[Image:KAH042815_gel1.jpg|250px|Hover name]]<br>15 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
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| DNA(plasmid) || 3.0 μL
| DNA(plasmid) || 3.0 μL
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{| {{table}} cellspacing="3" <!-- PCR rxn table -->
{| {{table}} cellspacing="3" <!-- PCR rxn table -->
| bgcolor=#cfcfcf || Reagent
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf || Vol
| bgcolor=#cfcfcf | Vol
| bgcolor=#cfcfcf || Mix (x2)
| bgcolor=#cfcfcf | Mix (x2)
| rowspan=7 || Expected:<br>1. pAubR = 153<br>2. pBjaR = 122<br>3. pBraR = 214<br>4. pRpaR = 136
| rowspan=7 | Expected:<br>1. pAubR = 153<br>2. pBjaR = 122<br>3. pBraR = 214<br>4. pRpaR = 136
| rowspan=7 | [[Image:KAH042815_gel2.jpg|250px|Hover name]]<br>5 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
|-
|-
| gBlock DNA (2 ng/μL) || 0.5  || 1.0  
| gBlock DNA (2 ng/μL) || 0.5  || 1.0  
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| dH<sub>2</sub>O    || 22.5  || 8.7  
| dH<sub>2</sub>O    || 22.5  || 8.7  
|-
|-
| &nbsp;            || 50.0 μL ||  ||
| &nbsp;            || 50.0 μL ||   
|}
|}


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* 72°C 3 min.
* 72°C 3 min.
* 4°C, ∞
* 4°C, ∞
* Digests (Fermentas FD)
** E/X
{| {{table}} cellspacing="3" <!-- Digest for dephos rxn. table -->
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume
|-
| DNA (20 ng) || 10.0 μL
|-
| 10x buffer || 2.0
|-
| EcoRI || 1.0
|-
| XbaI || 1.0
|-
| dH<sub>2</sub>O || 6.0
|-
| &nbsp; || 20.0 μL --> 37°C/ ~15 min.
|}
* Dephosphorylation (Roche)
** Assuming that shrimp alkaline phosphatase works in FastDigest buffer conditions [https://www.lifetechnologies.com/us/en/home/brands/thermo-scientific/molecular-biology/thermo-scientific-restriction-modifying-enzymes/modifying-enzymes-thermo-scientific/modifying-enzyme-buffers.html Link]
{| {{table}} cellspacing="3" <!-- Dephos table -->
|-
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume
|-
| DNA (digest) || 20.0 μL
|-
| phosphatase || 1.0
|-
| dH<sub>2</sub>O || ---
|-
| &nbsp; || 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
# 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 -->
| &nbsp;            || 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
|-
| dH<sub>2</sub>O    || 8.8  || 8.7  || 8.8
|-
| &nbsp;            || 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, ∞




Latest revision as of 00:56, 27 September 2017

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

04/28/15

  • Ryan - minipreps & digests
  • Ryan - PCR & Dig/Lig (promoter insert trial #2)


Minipreps

  • Use Sigma GenElute; elute with 75 μL elution solution
  • BraR/MRV colonies - picked 4 additional colonies from original cloning plate
  • Cut w/ E/X
  1. Bra/MRV 3
  2. Bra/MRV 4
  3. Bra/MRV 5
  4. Bra/MRV 6
  • promoter/Regulator/MRV colonies
  • Cut w/ KpnI/EcoRI - BbsI drop-in of promoter destroys KpnI site; empty vector will produce 2 bands, successful clones will be linearized (1 cut)
  1. pAub/AubR/MRV
  2. pBja/BjaR/MRV
  3. pRpa/RpaR/MRV


Reagent Volume Expected:
1-4. Bra/MRV = ~3200, 776
Empty MRV E/X = ~3200
5. pAub/AubR/MRV = 4240
6. pBja/BjaR/MRV = 4099
7. pRpa/RpaR/MRV = 4116
Empty vector = ~4000, 971 		Hover name
15 μL/lane; 1% agarose; Ladder
DNA(plasmid) 3.0 μL
10X buffer 1.5
enzyme 1 1.0
enzyme 2 1.0
dH2O 8.5
  15 μL --> 37°C/ ~15 min.


  • Measure [DNA]
Sample OD 260 260/280 ng/μL
BraR/MRV #5 0.465 1.913 465.4


Conclusions

  • BraR/MRV - Success! Keep colony 5 (lane 3)
  • promter/Reg/MRV - none worked. Try the assembly again with PCR-amplified promoter inserts


Ryan - PCR & Dig/Lig (promoter insert trial #2)

Ryan - Receiver plasmids, assembly - REVISED STRATEGY (PCR promoters)

  • 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
    • PCR-amplify promoter inserts (same primers as Stage 1)
    • 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
    • Values from last week, except BraR/MRV, which was measured today
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. BraR/MRV #5 0.465 1.913 465.4
4. RpaR/MRV 0.246 1.896 246.2
  • PCR-amplify promoters
    • 2x 50 μL rxns each
  1. pAubR, EcoRI fwd, XbaI rev
  2. pBjaR, EcoRI fwd, XbaI rev
  3. pBraR, EcoRI fwd, XbaI rev
  4. pRpaR, EcoRI fwd, XbaI rev
Reagent Vol Mix (x2) Expected:
1. pAubR = 153
2. pBjaR = 122
3. pBraR = 214
4. pRpaR = 136 		Hover name
5 μL/lane; 1% agarose; Ladder
gBlock DNA (2 ng/μL) 0.5 1.0
10 μM XbaI rev 1.0 2.0
10 μM EcoRI fwd 1.0 2.0
2x GoTaq green 25.0 50.0
dH2O 22.5 8.7
  50.0 μL

Thermal cycler: Labnet - GOTAQ

  • 95°C 3 min.
  • 30x[95°C, 30 sec; 57°C 30 sec; 72°C 30 sec]
  • 72°C 3 min.
  • 4°C, ∞




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