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

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(Autocreate 2015/04/17 Entry for User:Karmella_Haynes/Notebook/BioBrick_cloning)
 
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==mm/dd/yy==
==04/16/15==
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* Line item 1
* Rene - gRNA plasmids, 5 mL cultures (11 total)
* Line item 2
* Ryan - Receiver plasmids, assembly




----
----
'''Minipreps'''<br>
'''Ryan - Receiver plasmids, assembly'''<br>
* 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 (#/#). 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 -->
 
{| {{table}} border="1" cellspacing="3" <!-- Phusion PCR table -->
|- valign="top"
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Reagent
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| 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] -->
|-
|-
| DNA(plasmid) || 2.0 μL
| DNA(plasmid) || 0.5 μL
|-
|-
| 10X buffer || 1.5
| 5X HF buffer || 10.0
|-
|-
| EcoRI || 1.0
| EcoRI || 1.0

Revision as of 10:56, 17 April 2015

Karmella's BioBrick Cloning <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
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04/16/15

  • Rene - gRNA plasmids, 5 mL cultures (11 total)
  • Ryan - Receiver plasmids, assembly


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


Reagent Volume Expected:
1. BB 1 = size
2. BB2 = size
DNA(plasmid) 0.5 μL
5X HF buffer 10.0
EcoRI 1.0
PstI 1.0
dH2O 9.5
  15 μL --> 37°C/ ~15 min.

Assemblies

  1. BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
  2. BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size


  • Digests (Fermentas FD)
    • Specific notes
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.


  • 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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