User:Karmella Haynes/Notebook/BioBrick cloning/2015/04/21: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
Line 9: Line 9:
<!-- Precede finished items with a checkmark &#x2713; -->
<!-- Precede finished items with a checkmark &#x2713; -->
* Ryan - regulator assemblies
* Ryan - regulator assemblies
* Rene - check gRNA assemblies
* LCR Development - Phusion PCR
* LCR Development - Phusion PCR


Line 16: Line 17:
* Stage 1 - insert Regulator ORFS
* Stage 1 - insert Regulator ORFS
** DONE - Phusion PCR-amplify ORFs (add 5'-E, 3'-X cut sites)
** DONE - 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]
** '''Insert ORF(E/X) into Vector (E/X). see [https://benchling.com/s/VFNzuauP/edit Vector plasmid map]'''
* Stage 2 - insert Promoters
* Stage 2 - insert Promoters
** Cut & dephos promoters with E/X
** Cut & dephos promoters with E/X
Line 22: Line 23:




Clean up PCR products
* Clean up PCR products
* tba
** Zymo clean and concentrator
** Elute w/ 25 μL dH<sub>2</sub>O




Digests<br>
* Assemblies
* E/X
# AubR/MRV: AubRPCR(E/X)/851 + MRV(E/X)/size
# BjaR/MRV: BjaRPCR(E/X)/776 + MRV(E/X)/size
# BraR/MRV: BraRPCR(E/X)/776 + MRV(E/X)/size
# RpaR/MRV: RpaRPCR(E/X)/779 + MRV(E/X)/size
 
 
* Digests<br>
** E/X
# AubR, 851 bp
# AubR, 851 bp
# BjaR, 776 bp
# BjaR, 776 bp
# BraR, 776 bp
# BraR, 776 bp
# RpaR, 779 bp
# RpaR, 779 bp
# Receiver, Vector
# Modular Receiver Vector (MRV)


{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
Line 40: Line 49:
| bgcolor=#cfcfcf | Rxn 5
| bgcolor=#cfcfcf | Rxn 5
|-
|-
| DNA || 10.0 μL
| DNA || 10.0 || 5.0
|-
|-
| 10X buffer || 3.0
| 10X buffer || 3.0 || 3.0
|-
|-
| EcoRI || 1.0
| EcoRI || 1.0 || 1.0
|-
|-
| XbaI || 1.0
| XbaI || 1.0 || 1.0
|-
|-
| dH<sub>2</sub>O || 15.0
| dH<sub>2</sub>O || 15.0 || 20.0
|-
|-
| &nbsp; || 30 μL --> 37°C/ ~15 min.
| &nbsp; || 30.0 || 30.0
|}
|}






Column clean-up
* Column clean-up
* tba
** Zymo clean and concentrator
 
 


----
'''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)
** Specific notes
{| {{table}} cellspacing="3" <!-- Digest rxn. table -->
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| 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
|-
| 10x buffer || 3.0
|-
| enzyme 1 || 1.0
|-
| enzyme 2 || 1.0
|-
| dH<sub>2</sub>O || ---
|-
| &nbsp; || 30 μL --> 37°C/ ~30 min.
|}




Line 101: Line 77:
|-
|-
| 2. Digested part (c/d) || --- || --- || ---
| 2. Digested part (c/d) || --- || --- || ---
|}
* Dephosphorylation (Roche)
{| {{table}} cellspacing="3" <!-- Dephos table -->
|-
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume
|-
| DNA (clean digest) || up to 17 μL (500 ng)
|-
| 10x buffer d.p. || 2.0
|-
| phosphatase || 1.0
|-
| dH<sub>2</sub>O || ---
|-
| &nbsp; || 20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL
|}
|}


Line 147: Line 105:
| &nbsp;            || # μL || # μL ||
| &nbsp;            || # μL || # μL ||
|}
|}


----
----
'''Oligo annealing'''
'''Rene - check gRNA assemblies'''
# New BB 1
# New BB 2


{| class="wikitable" border="0" cellspacing="3" <!-- Oligo annealing rxn table -->
* Minipreps from 4/20/15
| DNA (oligos, 100 μM) || up to 18 μL (3 μL ea.)
** Note: ran out of time last week to grow up cultures. Stored at 4C until this week, grew for ~5 hours and then miniprepped w/ Sigma GenElute kit (elution vol. = 75 μL)
* Check w/ BpiI/XbaI
 
# gRNA51, 1
# gRNA51, 2
# gRNA52, 1
# gRNA52, 2
# gRNA53, 1
# gRNA53, 2
# gRNA54
# gRNA55, 1
# gRNA55, 2
# gRNA56, 1
# gRNA56, 2
# pX330 (ctrl)
 
 
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Rxn 1-11
| bgcolor=#cfcfcf | Mix(x13)
|-
|-
| 10x annealing buffer || 2.0
| DNA || 3.0 || ---
|-
|-
| dH<sub>2</sub>O || ---
| 10X buffer || 1.5 || 19.5
|-
|-
| &nbsp; || 20 μL --> 100°C (water bath)/ 5 min.; Cool to R.T. overnight
| BpiI || 0.5 || 6.5
|-
| XbaI || 0.5 || 6.5
|-
| dH<sub>2</sub>O || 9.5 || 123.5
|-
| &nbsp; || 15.0 ||
|}
|}




Revision as of 11:40, 21 April 2015

Karmella's BioBrick Cloning <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

04/21/15

  • Ryan - regulator assemblies
  • Rene - check gRNA assemblies
  • LCR Development - Phusion PCR


Ryan - Receiver plasmids, assembly

  • Stage 1 - insert Regulator ORFS
    • DONE - 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


  • Clean up PCR products
    • Zymo clean and concentrator
    • Elute w/ 25 μL dH2O


  • Assemblies
  1. AubR/MRV: AubRPCR(E/X)/851 + MRV(E/X)/size
  2. BjaR/MRV: BjaRPCR(E/X)/776 + MRV(E/X)/size
  3. BraR/MRV: BraRPCR(E/X)/776 + MRV(E/X)/size
  4. RpaR/MRV: RpaRPCR(E/X)/779 + MRV(E/X)/size


  • Digests
    • E/X
  1. AubR, 851 bp
  2. BjaR, 776 bp
  3. BraR, 776 bp
  4. RpaR, 779 bp
  5. Modular Receiver Vector (MRV)
Reagent Rxn 1-4 Rxn 5
DNA 10.0 5.0
10X buffer 3.0 3.0
EcoRI 1.0 1.0
XbaI 1.0 1.0
dH2O 15.0 20.0
  30.0 30.0


  • Column clean-up
    • Zymo clean and concentrator


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


  • 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


Rene - check gRNA assemblies

  • Minipreps from 4/20/15
    • Note: ran out of time last week to grow up cultures. Stored at 4C until this week, grew for ~5 hours and then miniprepped w/ Sigma GenElute kit (elution vol. = 75 μL)
  • Check w/ BpiI/XbaI
  1. gRNA51, 1
  2. gRNA51, 2
  3. gRNA52, 1
  4. gRNA52, 2
  5. gRNA53, 1
  6. gRNA53, 2
  7. gRNA54
  8. gRNA55, 1
  9. gRNA55, 2
  10. gRNA56, 1
  11. gRNA56, 2
  12. pX330 (ctrl)


Reagent Rxn 1-11 Mix(x13)
DNA 3.0 ---
10X buffer 1.5 19.5
BpiI 0.5 6.5
XbaI 0.5 6.5
dH2O 9.5 123.5
  15.0



Retrieved from "https://openwetware.org/mediawiki/index.php?title=User:Karmella_Haynes/Notebook/BioBrick_cloning/2015/04/21&oldid=881254"