User:Karmella Haynes/Notebook/BioBrick cloning/2015/05/26: Difference between revisions

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Line 54: Line 54:
* 72°C, 10 min
* 72°C, 10 min
* 4°C ∞
* 4°C ∞
* Column-clean
** Use Qiagen kit
** Elute & back-elute w/ 25 μL elution sln.




Line 59: Line 64:
** PCR, Clip & Clone protocol; digest w/ BsaI
** PCR, Clip & Clone protocol; digest w/ BsaI


* Column-clean
** Use Qiagen kit
** Elute & back-elute w/ 25 μL elution sln.




* Ligations
* Ligations
** DBN001 2-part backbone from [http://openwetware.org/wiki/User:Karmella_Haynes/Notebook/BioBrick_cloning/2015/05/15 05/15/15]
** DBN001 2-part backbone from [http://openwetware.org/wiki/User:Karmella_Haynes/Notebook/BioBrick_cloning/2015/05/15 05/15/15]
** 2:1 ratio calc.: 1972 bp insert /  
** 2:1 ratio calc.: 1972 bp insert / 2336 bp vector * 2 * 50 = '''84 ng insert'''
# DBN006(BsaI)/1972 + DBN001(BsaI)/2336
# DBN006/(BsaI)/1972 + DBN001/(BsaI)/2336
# DBN001(BsaI)/2336
# DBN001/(BsaI)/2336
 
{| {{table}} cellspacing="3" <!-- Ligation rxn table -->
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Rxn1
| bgcolor=#cfcfcf | Rxn2
|-
| Insert DNA        || 3.5  || ---
|-
| Vector DNA        || 3.0  || 3.0
|-
| 2x lgn buf (Roche) || 7.5  || 7.5
|-
| T4 ligase (NEB)    || 1.0  || 1.0
|-
| dH<sub>2</sub>O    || ---  || 3.5
|-
| &nbsp;            || 15.0 μL || 15.0 μL
|}
 




Revision as of 12:35, 26 May 2015

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05/26/15

  • Ben - luc replacement donor plasmid DBN007
  • Rene - CRISPR PCR library PCR trial


Ben - luc replacement donor plasmid

  • Assembly:
  1. DBN007_pSB1A3: DBN006(BsaI)/1972 + DBN001(BsaI)/1267+1069 (=2336)


Reagent Rxn1,2 Expected:
1,2. DBN006 = 1972 		Hover name
10 μL/lane, 1% agarose; Ladder
Template 0.2
10 uM fwd primer 1.0
10 uM rev primer 1.0
10 mM dNTPs 1.0
Phusion pol. 0.5
5x HF buffer 5.0
dH2O 41.3
  50.0

Bio-Rad: "Phusion" (block #)

  • 98°C, 3 min
  • 30x[98°C, 10 sec; 67°C, 30 sec; 72°C, 30 sec]
  • 72°C, 10 min
  • 4°C ∞


  • Column-clean
    • Use Qiagen kit
    • Elute & back-elute w/ 25 μL elution sln.


  • Digest & dephos
    • PCR, Clip & Clone protocol; digest w/ BsaI


  • Ligations
    • DBN001 2-part backbone from 05/15/15
    • 2:1 ratio calc.: 1972 bp insert / 2336 bp vector * 2 * 50 = 84 ng insert
  1. DBN006/(BsaI)/1972 + DBN001/(BsaI)/2336
  2. DBN001/(BsaI)/2336
Reagent Rxn1 Rxn2
Insert DNA 3.5 ---
Vector DNA 3.0 3.0
2x lgn buf (Roche) 7.5 7.5
T4 ligase (NEB) 1.0 1.0
dH2O --- 3.5
  15.0 μL 15.0 μL



Line item

  • Minipreps
    • Sigma GenElute kit, elute w/ 75 μL elution sln.
  • Digests
    • Check with #/# digests
Reagent Volume Expected:
1. BB 1 = size
2. BB2 = size
DNA(plasmid) 2.0 μL
10X buffer 1.5
EcoRI 1.0
PstI 1.0
dH2O 9.5
  15 μL --> 37°C/ ~15 min.

Line item


  • 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
  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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