User:Karmella Haynes/Notebook/BioBrick cloning/2015/05/21: Difference between revisions

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'''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 "MV11" (S/N)'''
* STAGE 4 - gRNA
** Put pre-existing gRNA (from luc experiment) into pSPgRNA
'''Assemblies'''
'''Assemblies'''
# H3-1-40_MV11: H3-1-40/(a/b)/size + 3' part/(c/d)/size
# H3-1-40_MV11: H3-1-40/(X/N)/size + MV11/(S/N)/9506
# H3-1-60_MV11: 5' part/(a/b)/size + 3' part/(c/d)/size
# H3-1-60_MV11: 5' part/(a/b)/size + MV11/(S/N)/9506




* Digests (Fermentas FD)
* Digest of vector (Fermentas FD)
** Specific notes
** SpeI/NotI


{| {{table}} cellspacing="3" <!-- Digest rxn. table -->
{| {{table}} cellspacing="3" <!-- Digest rxn. table -->
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| &nbsp; || 30 μL --> 37°C/ ~30 min.
| &nbsp; || 30 μL --> 37°C/ ~30 min.
|}
|}
* Digest of PCR fragments (PCR, Clip, & Clone)
** DpnI (to get rid of methylated vector)/XbaI/NotI




Revision as of 15:10, 21 May 2015

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05/21/15

  • Cas-tone - Assemblies, H3's + MV11


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 "MV11" (S/N)
  • STAGE 4 - gRNA
    • Put pre-existing gRNA (from luc experiment) into pSPgRNA


Assemblies

  1. H3-1-40_MV11: H3-1-40/(X/N)/size + MV11/(S/N)/9506
  2. H3-1-60_MV11: 5' part/(a/b)/size + MV11/(S/N)/9506


  • Digest of vector (Fermentas FD)
    • SpeI/NotI
Reagent Volume
DNA (plasmid) up to 25 μL
10x buffer 3.0
enzyme 1 1.0
enzyme 2 1.0
dH2O ---
  30 μL --> 37°C/ ~30 min.


  • Digest of PCR fragments (PCR, Clip, & Clone)
    • DpnI (to get rid of methylated vector)/XbaI/NotI



  • Measure conc.'s
Sample OD260 260/280 ng/μL
1. Digested part (a/b) --- --- ---
2. Digested part (c/d) --- --- ---


  • Dephosphorylation (Roche)
Reagent Volume
DNA (clean digest) up to 17 μL (500 ng)
10x buffer d.p. 2.0
phosphatase 1.0
dH2O ---
  20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL


  • Ligations
Ligation Plate results (lig : neg crtl) mm/dd/yy
1. insert(a/b)/size, ## ng + vector(c/d)/size, ## ng new BioBrick #:1 (Pick #)
2. vector(c/d)/ ## ng  
  1 2
Insert DNA ### ---
Vector DNA ### ###
2x lgn buf (Roche) ### ###
T4 ligase (NEB) 1.0 1.0
dH2O ### ###
  # μL # μL

Oligo annealing

  1. New BB 1
  2. New BB 2
DNA (oligos, 100 μM) up to 18 μL (3 μL ea.)
10x annealing buffer 2.0
dH2O ---
  20 μL --> 100°C (water bath)/ 5 min.; Cool to R.T. overnight



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