User:Karmella Haynes/Notebook/BioBrick cloning/2015/05/28: Difference between revisions

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* Assemblies
* Assemblies
# DT001 hPCD(E/S)/186 + PLflex_pUC57(E/X)/2763
# DT001_pUC57: hPCD(E/S)/186 + PLflex_pUC57(E/X)/2763
# DT002 hPCD(E/S)/186 + PLflex4_pUC57(E/X)/2930
# DT002_pUC57: hPCD(E/S)/186 + PLflex4_pUC57(E/X)/2930
# DT003 hPCD(E/S)/186 + PLrigid_pUC57(E/X)/
# DT003_pUC57: hPCD(E/S)/186 + PLrigid_pUC57(E/X)/2763
# DT004 hPCD(E/S)/186 + PLrigid4_pUC57(E/X)/
# DT004_pUC57: hPCD(E/S)/186 + PLrigid4_pUC57(E/X)/2931
 




* Digests (Fermentas FD)
* Digests (Fermentas FD)
** Specific notes


{| {{table}} cellspacing="3" <!-- Digest rxn. table -->
{| {{table}} cellspacing="3" <!-- Digest rxn. table -->
|- valign="top"
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume
| bgcolor=#cfcfcf | Rxn1
| 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] -->
| bgcolor=#cfcfcf | Rxn2-5
| rowspan="7" | Expected bands:<br>1. hPCD(E/S) = 186<br>2. PLflex_pUC57(E/X) = 2763<br>3. PLflex4_pUC57(E/X) = 2930<br>4. PLrigid_pUC57(E/X) = 2763<br>5. PLrigid4_pUC57(E/X) = 2931
| rowspan="7" | [[Image:somegel.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
| DNA (plasmid) || 25.0 || 15.0
|-
|-
| 10x buffer || 3.0
| 10x buffer || 3.0 || 3.0
|-
|-
| enzyme 1 || 1.0
| enzyme 1 || 1.0 || 1.0
|-
|-
| enzyme 2 || 1.0
| enzyme 2 || 1.0 || 1.0
|-
|-
| dH<sub>2</sub>O || ---
| dH<sub>2</sub>O || --- || 10.0
|-
|-
| &nbsp; || 30 μL --> 37°C/ ~30 min.
| &nbsp; || 30 μL  
|}
|}
--> 37°C/ ~30 min.
* Gel purification
** Sigma GenElute Gel purification kit
** Elute & back-elute w/ 25 μL elution sln.




Line 50: Line 59:
| Sample || OD260 || 260/280 || ng/μL
| Sample || OD260 || 260/280 || ng/μL
|-
|-
| 1. Digested part (a/b) || --- || --- || ---
| 1. hPCD(E/S) || --- || --- || ---
|-
|-
| 2. Digested part (c/d) || --- || --- || ---
| 2. PLflex_pUC57(E/X) || --- || --- || ---
|}
|}


* 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
|}




Revision as of 17:26, 28 May 2015

Karmella's BioBrick Cloning <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
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05/28/15

  • David Tze - assemblies: linkers + hPCD


David Tze - assemblies: linkers + hPCD

  • Assemblies
  1. DT001_pUC57: hPCD(E/S)/186 + PLflex_pUC57(E/X)/2763
  2. DT002_pUC57: hPCD(E/S)/186 + PLflex4_pUC57(E/X)/2930
  3. DT003_pUC57: hPCD(E/S)/186 + PLrigid_pUC57(E/X)/2763
  4. DT004_pUC57: hPCD(E/S)/186 + PLrigid4_pUC57(E/X)/2931


  • Digests (Fermentas FD)
Reagent Rxn1 Rxn2-5 Expected bands:
1. hPCD(E/S) = 186
2. PLflex_pUC57(E/X) = 2763
3. PLflex4_pUC57(E/X) = 2930
4. PLrigid_pUC57(E/X) = 2763
5. PLrigid4_pUC57(E/X) = 2931 		Hover name
30 μL/lane, 1% agarose; Ladder
DNA (plasmid) 25.0 15.0
10x buffer 3.0 3.0
enzyme 1 1.0 1.0
enzyme 2 1.0 1.0
dH2O --- 10.0
  30 μL

--> 37°C/ ~30 min.


  • Gel purification
    • Sigma GenElute Gel purification kit
    • Elute & back-elute w/ 25 μL elution sln.


  • Measure conc.'s
Sample OD260 260/280 ng/μL
1. hPCD(E/S) --- --- ---
2. PLflex_pUC57(E/X) --- --- ---


  • Ligations
  1. insert/(a/b)/size + vector/(c/d)/size
  2. vector/(c/d)/size
Reagent Rxn1 Rxn2
Insert DNA ### ---
Vector DNA ### ###
2x lgn buf (Roche) ### ###
T4 ligase (NEB) 1.0 1.0
dH2O ### ###
  # μL # μL




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