User:Karmella Haynes/Notebook/BioBrick cloning/2015/05/22: Difference between revisions

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| dH<sub>2</sub>O || 10.5
| dH<sub>2</sub>O || 10.5
|-
|-
| &nbsp; || 15 μL --> 37°C/ ~15 min.
| &nbsp; || 15 μL  
|}
|}
 
--> 37°C/ ~15 min.




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* Minipreps<br>
** Check with S/HindIII digests - H3 insertion destroys SpeI site
** Sigma GenElute kit, elute w/ 75 μL elution sln.
** Note: Fragment without H3 part = 7162; added H3-1-# size + 7162 to get lengths for longer H/H band
 
* Digests
** Check with ### digests - promoter insertion generates a new EcoRI site
** Note: BraR = 776, pBra = 214


{| {{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>1-8. pBra-BraR_MRV = 2989, 1202<br>empty vector = 3977<br>
| rowspan="7" | <u>Expected:</u><br>1,2. H3-1-40_MV11 = 2345, 7161+##<br>3,4. H3-1-60_MV11 = 2345, 7162+##<br>5,6. H3-1-80_MV11 = 2345, 7162+##<br>7,8. H3-1-116_MV11 = 2345, 7162+##<br>9,10. H3-1-136_MV11 = 2345, 7162+##<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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| 10X buffer || 1.5
| 10X buffer || 1.5
|-
|-
| EcoRI || 1.0
| SpeI || 1.0
|-
| HindIII || 1.0
|-
|-
| dH<sub>2</sub>O || 10.5
| dH<sub>2</sub>O || 9.5
|-
|-
| &nbsp; || 15 μL --> 37°C/ ~15 min.
| &nbsp; || 15 μL  
|}
|}
--> 37°C/ ~15 min.




Revision as of 11:20, 22 May 2015

Karmella's BioBrick Cloning <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
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05/22/15

  • Ryan - Bra minipreps & digests
  • Cas-tone - minipreps & digests
  • Ben - luc replacement donor plasmid


Ryan - Bra minipreps & digests

  • Minipreps
    • Sigma GenElute kit, elute w/ 75 μL elution sln.
  • Digests
    • Check with EcoRI digests - promoter insertion generates a new EcoRI site
    • Note: BraR = 776, pBra = 214
Reagent Volume Expected:
1-8. pBra-BraR_MRV = 2989, 1202
empty vector = 3977
DNA(plasmid) 2.0 μL
10X buffer 1.5
EcoRI 1.0
dH2O 10.5
  15 μL

--> 37°C/ ~15 min.


Cas-tone - H3_MV11 minipreps & digests

  • Cultures
    • Pick 2 colonies from each ligation plate
    • Make streak library & 5 mL cultures


    • Check with S/HindIII digests - H3 insertion destroys SpeI site
    • Note: Fragment without H3 part = 7162; added H3-1-# size + 7162 to get lengths for longer H/H band
Reagent Volume Expected:
1,2. H3-1-40_MV11 = 2345, 7161+##
3,4. H3-1-60_MV11 = 2345, 7162+##
5,6. H3-1-80_MV11 = 2345, 7162+##
7,8. H3-1-116_MV11 = 2345, 7162+##
9,10. H3-1-136_MV11 = 2345, 7162+##
DNA(plasmid) 2.0 μL
10X buffer 1.5
SpeI 1.0
HindIII 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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