User:Karmella Haynes/Notebook/BioBrick cloning/2015/05/19: Difference between revisions

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'''Minipreps'''<br>
'''Minipreps'''<br>
* Check with E/P digests
* Check with E/P digests
** Modification (dsOligo insert replacing VP64) removes an EcoRI site


{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
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| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume  
| bgcolor=#cfcfcf | Volume  
| rowspan="7" | <u>Expected:</u><br>1. BB 1 = size<br>2. BB2 = size<br>
| rowspan="7" | <u>Expected:</u><br>1,2. CMV-dCas9-HA = 5317, 4189<br>CMV-dCas9-VP64 =5459, 3016, 1337<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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| dH<sub>2</sub>O || 9.5
| dH<sub>2</sub>O || 9.5
|-
|-
| &nbsp; || 15 μL --> 37°C/ ~15 min.
| &nbsp; || 15 μL  
|}
|}
--> 37°C/ ~10 min.


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Revision as of 13:35, 19 May 2015

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05/19/15

  • Cas-tone - cultures, minipreps, digests
  • Rene - PCR library 96-well plates


Minipreps

  • Check with E/P digests
    • Modification (dsOligo insert replacing VP64) removes an EcoRI site
Reagent Volume Expected:
1,2. CMV-dCas9-HA = 5317, 4189
CMV-dCas9-VP64 =5459, 3016, 1337
DNA(plasmid) 2.0 μL
10X buffer 1.5
EcoRI 1.0
PstI 1.0
dH2O 9.5
  15 μL

--> 37°C/ ~10 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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