User:Karmella Haynes/Notebook/BioBrick cloning/2015/10/08: 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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==mm/dd/yy==
==10/09/15==
<!-- Precede finished items with a checkmark &#x2713; -->
<!-- Precede finished items with a checkmark &#x2713; -->
* Line item 1
* Cas-tone- H3 inserts into MV12 (new MV11-GFP)
* Line item 2




----
----
'''Line item'''
'''Cas-tone Project'''
* STAGE 1 - pcDNA-dCas9-VP64 vector re-design
** '''DONE''' - Knock-out VP64 from C-terminus - replace AscI-VP64:HA:STOP-EcoRI-XbaI with AscI-HA:STOP-XbaI dsOligo
** '''DONE''' - Knock-out FLAG from N-terminus - replace SpeI-CMV:3xFLAG:NLS-SacII with PCR-amplified (SpeI)/XbaI-CMV-SpeI-NotI-SacII; use reverse primer to add SacII
* STAGE 2 - Histone parts
** '''DONE''' - Order primers (annotated in Benchling)
** '''DONE''' PCR-amplify histone parts as XbaI-Kozak-histone part-a-NotI. Note: Do no use Kozak BioBrick, since this will add extra amino acids onto conserved histone N-terminal tail. Instead, use the Kozak-like seq. from the Addgene plasmid, +3 extra Kozak bases (included in primer)
* STAGE 3 - Cas-fusion
** '''Insert Kozak-histone parts (X/N) into dCas9 plasmid + GFP reporter "MV12" (S/N)'''
* STAGE 4 - gRNA
** Put pre-existing gRNA (from luc experiment) into pSPgRNA


* Minipreps
** Sigma GenElute kit, elute w/ 75 μL elution sln.


* Digests
'''Assemblies'''
** Check with #/# digests
# H3-1-40_MV12: H3-1-40/(X/N)/129 + '''MV12/(S/N)/10305'''
 
# H3-1-60_MV12: H3-1-60/(X/N)/189 + "
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
# H3-1-80_MV12: H3-1-80/(X/N)/250 + "
|- valign="top"
# H3-1-116_MV12: H3-1-116/(X/N)/373 + "
| bgcolor=#cfcfcf | Reagent
# H3-1-136_MV12: H3-1-136/(X/N)/433 + "
| bgcolor=#cfcfcf | Volume
| rowspan="7" | <u>Expected:</u><br>1. BB 1 = size<br>2. BB2 = size<br>
| 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] -->
|-
| DNA(plasmid) || 2.0 μL
|-
| 10X buffer || 1.5
|-
| EcoRI || 1.0
|-
| PstI || 1.0
|-
| dH<sub>2</sub>O || 9.5
|-
| &nbsp; || 15 μL --> 37°C/ ~15 min.
|}
 
----
'''Line item'''
 
 
* 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)
* Digest of vector (Fermentas FD)
** Specific notes
** SpeI/NotI


{| {{table}} cellspacing="3" <!-- Digest rxn. table -->
{| {{table}} cellspacing="3" <!-- Digest rxn. table -->
Line 59: Line 42:
| 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) || 25.0
|-
|-
| 10x buffer || 3.0
| 10x buffer || 3.0
|-
|-
| enzyme 1 || 1.0
| SpeI || 1.0
|-
|-
| enzyme 2 || 1.0
| NotI || 1.0
|-
|-
| dH<sub>2</sub>O || ---
| dH<sub>2</sub>O || ---
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| &nbsp; || 30 μL --> 37°C/ ~30 min.
| &nbsp; || 30 μL --> 37°C/ ~30 min.
|}
|}
* Gel purification
** Sigma GenElute kit. Elute & back elute w/ 30 μL elution sln.




* Measure conc.'s
 
* DpnI digest & clean-up of PCR products
** Done on [http://openwetware.org/wiki/User:Karmella_Haynes/Notebook/BioBrick_cloning/2015/06/23 06/23/15]
 
 
* Measure [DNA] for vector and inserts
{| {{table}} cellspacing="3" <!-- [DNA] table -->
{| {{table}} cellspacing="3" <!-- [DNA] table -->
|- bgcolor=#cfcfcf  
|- bgcolor=#cfcfcf  
| Sample || OD260 || 260/280 || ng/μL
| Sample || OD260 || 260/280 || ng/μL
|-
|-
| 1. Digested part (a/b) || --- || --- || ---
| 1. MV11 (S/N) || 0.024 || 1.746 || 24.15
|-
| 2. Digested part (c/d) || --- || --- || ---
|}
 
 
* Dephosphorylation (Roche)
{| {{table}} cellspacing="3" <!-- Dephos table -->
|-
|-
| bgcolor=#cfcfcf | Reagent
| 2. H3-1-40 PCR || 0.03 || 1.879 || 29.8 [http://openwetware.org/wiki/User:Karmella_Haynes/Notebook/BioBrick_cloning/2015/06/23 06/23/15]
| bgcolor=#cfcfcf | Volume
|-
|-
| DNA (clean digest) || up to 17 μL (500 ng)
| 3. H3-1-60 PCR || 0.04 || 1.974 || 39.7 [http://openwetware.org/wiki/User:Karmella_Haynes/Notebook/BioBrick_cloning/2015/06/23 06/23/15]
|-
|-
| 10x buffer d.p. || 2.0
| 4. H3-1-80 PCR || 0.047 || 1.865 || 46.8 [http://openwetware.org/wiki/User:Karmella_Haynes/Notebook/BioBrick_cloning/2015/06/23 06/23/15]
|-
|-
| phosphatase || 1.0
| 5. H3-1-116 PCR || 0.06 || 1.887 || 59.57 [http://openwetware.org/wiki/User:Karmella_Haynes/Notebook/BioBrick_cloning/2015/06/23 06/23/15]
|-
|-
| dH<sub>2</sub>O || ---
| 6. H3-1-136 PCR || 0.062 || 1.932 || 62.0 [http://openwetware.org/wiki/User:Karmella_Haynes/Notebook/BioBrick_cloning/2015/06/23 06/23/15]
|-
| &nbsp; || 20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL
|}
|}




* Ligations
* Digest & dephos inserts
# insert/(a/b)/size + vector/(c/d)/size
** Done on [http://openwetware.org/wiki/User:Karmella_Haynes/Notebook/BioBrick_cloning/2015/06/23 06/23/15]
# vector/(c/d)/size
** Final concentration = 25 ng/μL.
 
 
 
* Ligations (10/12/15)
** > 2:1 ratio calculations...
** 433 bp largest insert / 10305 bp vector * 2 * 50 ng vector = 4.2 ng
** 129 bp smallest insert / 10305 bp vector * 2 * 50 ng vector = 1.3 ng


{| {{table}} cellspacing="3" <!-- Ligation rxn table -->
{| {{table}} cellspacing="3" <!-- Ligation rxn table -->
|- valign="top"
|- bgcolor="#cfcfcf" valign="top"
| bgcolor=#cfcfcf | Reagent
| Reagent
| bgcolor=#cfcfcf | Rxn1
| Rxn1-5
| bgcolor=#cfcfcf | Rxn2
| Rxn6
|-
|-
| Insert DNA        || ### || ---  
| Insert DNA        || 0.5 || ---
|-
|-
| Vector DNA        || ### || ###
| Vector DNA        || 2.0 || 2.0 
|-
|-
| 2x lgn buf (Roche) || ### || ###
| 2x lgn buf (Roche) || 5.0 || 5.0
|-
|-
| T4 ligase (NEB)    || 1.0  || 1.0  
| T4 ligase (NEB)    || 1.0  || 1.0
|-
|-
| dH<sub>2</sub>O    || ### || ###
| dH<sub>2</sub>O    || 1.5 || 2.0
|-
|-
| &nbsp;            || # μL || # μL
| &nbsp;            || 10.0 μL || 10.0 μL  
|}
|}


RESULTS (10/13/15)
* tba




Latest revision as of 01:14, 27 September 2017

Karmella's BioBrick Cloning Main project page
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10/09/15

  • Cas-tone- H3 inserts into MV12 (new MV11-GFP)


Cas-tone Project

  • STAGE 1 - pcDNA-dCas9-VP64 vector re-design
    • DONE - Knock-out VP64 from C-terminus - replace AscI-VP64:HA:STOP-EcoRI-XbaI with AscI-HA:STOP-XbaI dsOligo
    • DONE - Knock-out FLAG from N-terminus - replace SpeI-CMV:3xFLAG:NLS-SacII with PCR-amplified (SpeI)/XbaI-CMV-SpeI-NotI-SacII; use reverse primer to add SacII
  • STAGE 2 - Histone parts
    • DONE - Order primers (annotated in Benchling)
    • DONE PCR-amplify histone parts as XbaI-Kozak-histone part-a-NotI. Note: Do no use Kozak BioBrick, since this will add extra amino acids onto conserved histone N-terminal tail. Instead, use the Kozak-like seq. from the Addgene plasmid, +3 extra Kozak bases (included in primer)
  • STAGE 3 - Cas-fusion
    • Insert Kozak-histone parts (X/N) into dCas9 plasmid + GFP reporter "MV12" (S/N)
  • STAGE 4 - gRNA
    • Put pre-existing gRNA (from luc experiment) into pSPgRNA


Assemblies

  1. H3-1-40_MV12: H3-1-40/(X/N)/129 + MV12/(S/N)/10305
  2. H3-1-60_MV12: H3-1-60/(X/N)/189 + "
  3. H3-1-80_MV12: H3-1-80/(X/N)/250 + "
  4. H3-1-116_MV12: H3-1-116/(X/N)/373 + "
  5. H3-1-136_MV12: H3-1-136/(X/N)/433 + "


  • Digest of vector (Fermentas FD)
    • SpeI/NotI
Reagent Volume
DNA (plasmid) 25.0
10x buffer 3.0
SpeI 1.0
NotI 1.0
dH2O ---
  30 μL --> 37°C/ ~30 min.
  • Gel purification
    • Sigma GenElute kit. Elute & back elute w/ 30 μL elution sln.


  • DpnI digest & clean-up of PCR products


  • Measure [DNA] for vector and inserts
Sample OD260 260/280 ng/μL
1. MV11 (S/N) 0.024 1.746 24.15
2. H3-1-40 PCR 0.03 1.879 29.8 06/23/15
3. H3-1-60 PCR 0.04 1.974 39.7 06/23/15
4. H3-1-80 PCR 0.047 1.865 46.8 06/23/15
5. H3-1-116 PCR 0.06 1.887 59.57 06/23/15
6. H3-1-136 PCR 0.062 1.932 62.0 06/23/15


  • Digest & dephos inserts
    • Done on 06/23/15
    • Final concentration = 25 ng/μL.


  • Ligations (10/12/15)
    • > 2:1 ratio calculations...
    • 433 bp largest insert / 10305 bp vector * 2 * 50 ng vector = 4.2 ng
    • 129 bp smallest insert / 10305 bp vector * 2 * 50 ng vector = 1.3 ng
Reagent Rxn1-5 Rxn6
Insert DNA 0.5 ---
Vector DNA 2.0 2.0
2x lgn buf (Roche) 5.0 5.0
T4 ligase (NEB) 1.0 1.0
dH2O 1.5 2.0
  10.0 μL 10.0 μL

RESULTS (10/13/15)

  • tba



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