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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<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
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==mm/dd/yy==
==04/23/15==
<!-- Precede finished items with a checkmark &#x2713; -->
<!-- Precede finished items with a checkmark &#x2713; -->
* Line item 1
* Ryan - Receiver cloning stage 2
* Line item 2
* Rene - sequencing for g053-2, g054




----
----
'''Minipreps'''<br>
'''Ryan - Receiver plasmids, assembly'''
* Check with E/P digests
* 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 [https://benchling.com/s/VFNzuauP/edit 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'''


{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
 
|- valign="top"
'''Assemblies'''
| bgcolor=#cfcfcf | Reagent
# pAub/AubR/MRV: pAub(E/Xdp)/153 + AubR/MRV(BbsI)/4087
| bgcolor=#cfcfcf | Volume
# pBja/BjaR/MRV: pBja(E/Xdp)/122 + BjaR/MRV(BbsI)/3977
| rowspan="7" | <u>Expected:</u><br>1. BB 1 = size<br>2. BB2 = size<br>
# pBra/BraR/MRV: pBra(E/Xdp)/214 + BraR/MRV(BbsI)/3977
| 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] -->
# pRpa/RpaR/MRV: pRpa(E/Xdp)/136 + RpaR/MRV(BbsI)/3980
|-
 
| DNA(plasmid) || 2.0 μL
 
|-
* Measure conc.'s
| 10X buffer || 1.5
{| {{table}} cellspacing="3" <!-- [DNA] table -->
|-
|- bgcolor=#cfcfcf  
| EcoRI || 1.0
| Sample || OD260 || 260/280 || ng/μL
|-
|-
| PstI || 1.0
| 1. AubR/MRV || 0.258 || 1.906 || 257.7
|-
|-
| dH<sub>2</sub>O || 9.5
| 2. BjaR/MRV || 0.177 || 1.904 || 177.3
|-
|-
| &nbsp; || 15 μL --> 37°C/ ~15 min.
| 3. RpaR/MRV || 0.246 || 1.896 || 246.2
|}
|}
----
'''Assemblies'''
# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size




* Digests (Fermentas FD)
* Digests (Fermentas FD)
** Specific notes
** E/X
** Inserts are gBlocks = 2.0 ng/μL


{| {{table}} cellspacing="3" <!-- Digest rxn. table -->
{| {{table}} cellspacing="3" <!-- Digest for dephos rxn. table -->
|- valign="top"
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume
| bgcolor=#cfcfcf | Volume
| 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 (20 ng) || 10.0 μL
|-
|-
| 10x buffer || 3.0
| 10x buffer || 2.0
|-
|-
| enzyme 1 || 1.0
| EcoRI || 1.0
|-
|-
| enzyme 2 || 1.0
| XbaI || 1.0
|-
| dH<sub>2</sub>O || ---
|-
| &nbsp; || 30 μL --> 37°C/ ~30 min.
|}
 
 
* Measure conc.'s
{| {{table}} cellspacing="3" <!-- [DNA] table -->
|- bgcolor=#cfcfcf
| Sample || OD260 || 260/280 || ng/μL
|-
|-
| 1. Digested part (a/b) || --- || --- || ---
| dH<sub>2</sub>O || 6.0
|-
|-
| 2. Digested part (c/d) || --- || --- || ---
| &nbsp; || 20.0 μL --> 37°C/ ~15 min.
|}
|}




* Dephosphorylation (Roche)
* 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 -->
{| {{table}} cellspacing="3" <!-- Dephos table -->
|-
|-
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| bgcolor=#cfcfcf | Volume
| bgcolor=#cfcfcf | Volume
|-
|-
| DNA (clean digest) || up to 17 μL (500 ng)
| DNA (digest) || 20.0 μL (20 ng)
|-
| 10x buffer d.p. || 2.0
|-
|-
| phosphatase || 1.0
| phosphatase || 1.0
Line 90: Line 78:
| dH<sub>2</sub>O || ---
| dH<sub>2</sub>O || ---
|-
|-
| &nbsp; || 20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL
| &nbsp; || 20 μL  
|}
|}
--> 37°C/ 10 min.; 75°C/ 2 min.; [final] = ~1 ng/μL




* Ligations
 
{| {{table}} cellspacing="3" <!-- Ligations table -->
* Digestion/Ligation Reactions (borrowed from Rene's gRNA/Cas9 assembly procedure)
|- bgcolor=#cfcfcf
** Inserts are gBlocks = ~2ng/μL
| Ligation || <font color="blue"><u>Plate results (lig : neg crtl)</u> mm/dd/yy</font>
** 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   
|-
|-
| 1. insert(a/b)/size, ## ng + vector(c/d)/size, ## ng || <font color="blue">new BioBrick #:1 (Pick #)</font>
| Insert DNA        || 5.0  || 5.0  || 5.0
|-
|-
| 2. vector(c/d)/ ## ng || &nbsp;
| Vector DNA        || 0.2 || 0.3  || 0.2
|}
|-
 
| 10x FD buf        || 2.0  || 2.0  || 2.0
{| {{table}} cellspacing="3" <!-- Ligation rxn table -->
| &nbsp;            || 1    || 2   ||
|-
|-
| Insert DNA        || ###  || ---  ||
| 10 mM DTT          || 1.0 || 1.0 || 1.0
|-
|-
| Vector DNA        || ###  || ###  ||
| 10 mM ATP          || 1.0 || 1.0 || 1.0
|-
|-
| 2x lgn buf (Roche) || ### || ### ||
| FD BbsI/BpiI      || 1.0 || 1.0 || 1.0
|-
|-
| T4 ligase (NEB)   || 1.0  || 1.0  ||
| T4 ligase (Roche) || 1.0  || 1.0  || 1.0
|-
|-
| dH<sub>2</sub>O    || ### || ### ||
| dH<sub>2</sub>O    || 8.8 || 8.7 || 8.8
|-
|-
| &nbsp;            || # μL || # μL ||
| &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, ∞


----
----
'''Oligo annealing'''
'''Rene - sequencing'''
# New BB 1
 
# New BB 2
# g53-2, f (P139) - Confirmed
# g53-2, r (P140) - low Phred 20 score
# g54, f (P139) - Confirmed
# g54, r (P140) - low Phred 20 score
 
 


{| class="wikitable" border="0" cellspacing="3" <!-- Oligo annealing rxn table -->
| DNA (oligos, 100 μM) || up to 18 μL (3 μL ea.)
|-
| 10x annealing buffer || 2.0
|-
| dH<sub>2</sub>O || ---
|-
| &nbsp; || 20 μL --> 100°C (water bath)/ 5 min.; Cool to R.T. overnight
|}




Latest revision as of 00:55, 27 September 2017

Karmella's BioBrick Cloning Main project page
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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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