User:Karmella Haynes/Notebook/BioBrick cloning/2015/04/17: Difference between revisions

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* Rene - gRNA plasmids, 5 mL cultures (11 total)
* Rene - gRNA plasmids, 5 mL cultures (11 total)
* Ryan - Receiver plasmids, assembly
* Ryan - Receiver plasmids, assembly
* LCR Development - Repeat Phusion for MV10, use correct amount of 5x buffer




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Phusion PCR
Phusion PCR
#
# AubR, EcoRI F/ XbaI R
#
# BjaR, "
#
# BraR, "
#
# RpaR, "


{| {{table}} border="1" cellspacing="3" <!-- Phusion PCR table -->
{| {{table}} border="1" cellspacing="3" <!-- Phusion PCR table -->
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| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume  
| bgcolor=#cfcfcf | Volume  
| bgcolor=#cfcfcf | Mix (x#)
| bgcolor=#cfcfcf | Mix (x5)
| rowspan="7" | <u>Expected:</u><br>1. BB 1 = size<br>2. BB2 = size<br>
| 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] -->
| 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] -->
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| DNA(plasmid) || 0.5 μL || ---
| DNA(plasmid) || 0.5 μL || ---
|-
|-
| 10 μM F primer || 1.0 ||  
| 10 μM F primer || 1.0 || 5.0
|-
|-
| 10 μM R primer || 1.0 ||  
| 10 μM R primer || 1.0 || 5.0
|-
|-
| 10 mM dNTPs || 1.0 ||  
| 10 mM dNTPs || 1.0 || 5.0
|-
|-
| Phusion Pol. || 0.5 ||  
| Phusion Pol. || 0.5 || 2.5
|-
|-
| 5X HF buffer || 10.0 ||  
| 5X HF buffer || 10.0 || 50.0
|-
|-
| dH<sub>2</sub>O || 36.0 ||  
| dH<sub>2</sub>O || 36.0 || 180.0
|-
|-
| &nbsp; || 50.0 ||  
| &nbsp; || 50.0 ||  
|}
----
'''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.
|}
* Measure conc.'s
{| {{table}} cellspacing="3" <!-- [DNA] table -->
|- bgcolor=#cfcfcf
| Sample || OD260 || 260/280 || ng/μL
|-
| 1. Digested part (a/b) || --- || --- || ---
|-
| 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
|}
* Ligations
{| {{table}} cellspacing="3" <!-- Ligations table -->
|- bgcolor=#cfcfcf
| Ligation || <font color="blue"><u>Plate results (lig : neg crtl)</u> mm/dd/yy</font>
|-
| 1. insert(a/b)/size, ## ng + vector(c/d)/size, ## ng || <font color="blue">new BioBrick #:1 (Pick #)</font>
|-
| 2. vector(c/d)/ ## ng || &nbsp;
|}
{| {{table}} cellspacing="3" <!-- Ligation rxn table -->
| &nbsp;            || 1    || 2    ||
|-
| Insert DNA        || ###  || ---  ||
|-
| Vector DNA        || ###  || ###  ||
|-
| 2x lgn buf (Roche) || ###  || ###  ||
|-
| T4 ligase (NEB)    || 1.0  || 1.0  ||
|-
| dH<sub>2</sub>O    || ###  || ###  ||
|-
| &nbsp;            || # μL || # μL ||
|}
----
'''Oligo annealing'''
# New BB 1
# New BB 2
{| 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
|}
|}


Revision as of 12:01, 17 April 2015

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04/16/15

  • Rene - gRNA plasmids, 5 mL cultures (11 total)
  • Ryan - Receiver plasmids, assembly
  • LCR Development - Repeat Phusion for MV10, use correct amount of 5x buffer


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 (#/#). 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


Phusion PCR

  1. AubR, EcoRI F/ XbaI R
  2. BjaR, "
  3. BraR, "
  4. RpaR, "
Reagent Volume Mix (x5) Expected:
1. BB 1 = size
2. BB2 = size
DNA(plasmid) 0.5 μL ---
10 μM F primer 1.0 5.0
10 μM R primer 1.0 5.0
10 mM dNTPs 1.0 5.0
Phusion Pol. 0.5 2.5
5X HF buffer 10.0 50.0
dH2O 36.0 180.0
  50.0



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