User:Karmella Haynes/Notebook/BioBrick cloning/2012/12/11: Difference between revisions

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(Autocreate 2012/12/11 Entry for User:Karmella_Haynes/Notebook/BioBrick_cloning)
 
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==mm/dd/yy==
==12/11/12==
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* Line item 1
* Assembly: KAH111 + C-His (trouble shooting for Behzad, start from plasmids)
* Line item 2
* Minipreps: Rene's pSB1A3-RFP (2); cut with X/S for Brady and Vi's Gibson cloning




----
----
'''Minipreps'''<br>
'''Minipreps'''<br>
* Check with E/P digests
* pSB1A3-RFP (retransformation from 12/10/12)
* Concentrations: (1) 260 = , 260/280 = , '''ng/μL = '''; (2) 260 = , 260/280 = , '''ng/μL = ''';
* Digest 10 μL of each with X/S for Gibson cloning (Brady and Vi)


{| {{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>3. pSB1A3 = size<br>4. 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:KAH121112_gel1.png|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) || 10.0 μL
|-
|-
| 10X buffer || 1.5
| 10X buffer || 1.5

Revision as of 16:20, 11 December 2012

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12/11/12

  • Assembly: KAH111 + C-His (trouble shooting for Behzad, start from plasmids)
  • Minipreps: Rene's pSB1A3-RFP (2); cut with X/S for Brady and Vi's Gibson cloning


Minipreps

  • pSB1A3-RFP (retransformation from 12/10/12)
  • Concentrations: (1) 260 = , 260/280 = , ng/μL = ; (2) 260 = , 260/280 = , ng/μL = ;
  • Digest 10 μL of each with X/S for Gibson cloning (Brady and Vi)
Reagent Volume Expected:
3. pSB1A3 = size
4. BB2 = size
 Hover name
15 μL/lane; 1% agarose; Ladder
DNA(plasmid) 10.0 μL
10X buffer 1.5
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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