User:Karmella Haynes/Notebook/BioBrick cloning/2013/01/06: Difference between revisions

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(Autocreate 2013/01/06 Entry for User:Karmella_Haynes/Notebook/BioBrick_cloning)
 
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==mm/dd/yy==
==01/06/13==
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* Line item 1
* Gibson Asembly: pick colonies & check via colony PCR
* Line item 2
* Media: Make LB agar +amp




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----
'''Minipreps'''<br>
'''Gibson Assembly results'''<br>
* Check with E/P digests
* Plates from [http://openwetware.org/index.php?title=User:Karmella_Haynes/Notebook/BioBrick_cloning/2013/01/05 01/05/2013]


{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
* hPCD + BL01 + V0120, 2μL 1x Gibson: no colonies for assembly (1) or no-insert control (2)
|- valign="top"
* hPCD + BL01 + pSB1A3, 2μL 1x Gibson: 1 colony for assembly (3), none on control (4)
* hPCD + BL01 + V0120, 2μL 1/4x Gibson: 2 colonies for assembly (5), 1 on control (6)
* hPCD + BL01 + pSB1A3, 2μL 1/4x Gibson: 1 colony for assembly (7), 2 on control (8)
 
* Pick and streak colonies from plates 3, 5, 7
* Use same pipette tip for colony PCR
 
{| {{table}} cellspacing="3" <!-- Dephos table -->
|-
| 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>
| bgcolor=#cfcfcf | 5x mix
| 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
| Colony || --- || ---
|-
|-
| 10X buffer || 1.5
| 10 μM primer BL9 || 1.0 || 5.0
|-
|-
| EcoRI || 1.0
| 10 μM primer BL10 || 1.0 || 5.0
|-
|-
| PstI || 1.0
| 2x GoTaq || 12.5 || 62.5
|-
|-
| dH<sub>2</sub>O || 9.5
| dH<sub>2</sub>O || 10.5 || 52.5
|-
|-
| &nbsp; || 15 μL --> 37°C/ ~15 min.
| &nbsp; || 25.0 μL ||
|}
|}
PCR
* 95°C, 3 min.
* [95°C, 30 sec.; 95°C, 30 sec.; 72°C, 1 min.] x35
* 72°C, 3 min.
* 4°C, ∞


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Revision as of 12:20, 6 January 2013

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01/06/13

  • Gibson Asembly: pick colonies & check via colony PCR
  • Media: Make LB agar +amp


Gibson Assembly results

  • hPCD + BL01 + V0120, 2μL 1x Gibson: no colonies for assembly (1) or no-insert control (2)
  • hPCD + BL01 + pSB1A3, 2μL 1x Gibson: 1 colony for assembly (3), none on control (4)
  • hPCD + BL01 + V0120, 2μL 1/4x Gibson: 2 colonies for assembly (5), 1 on control (6)
  • hPCD + BL01 + pSB1A3, 2μL 1/4x Gibson: 1 colony for assembly (7), 2 on control (8)
  • Pick and streak colonies from plates 3, 5, 7
  • Use same pipette tip for colony PCR
Reagent Volume 5x mix
Colony --- ---
10 μM primer BL9 1.0 5.0
10 μM primer BL10 1.0 5.0
2x GoTaq 12.5 62.5
dH2O 10.5 52.5
  25.0 μL

PCR

  • 95°C, 3 min.
  • [95°C, 30 sec.; 95°C, 30 sec.; 72°C, 1 min.] x35
  • 72°C, 3 min.
  • 4°C, ∞


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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