User:Karmella Haynes/Notebook/BioBrick cloning/2015/01/27: Difference between revisions

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'''PCR'''
'''PCR'''
* Use purified KAH60 fragments as template for PCR
* Use purified KAH60 fragments as template for PCR
# KAH60 "backbone" (without VP64, includes scars) = 6637: KAH60 F1/ KAH60 R1




{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
{| {{table}} cellspacing="3" <!-- PCR rxn. table -->
|- valign="top"
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume
| bgcolor=#cfcfcf | Rxn1
| rowspan="7" | <u>Expected:</u><br>1. BB 1 = size<br>2. BB2 = size<br>
| rowspan="7" | Expected:<br>1. KAH60 = 6637
| 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:KAH012315_gel.jpg|270px|Hover name]]<br>10 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
|-
|-
| DNA(plasmid) || 2.0 μL
| Template || 0.2  
|-
|-
| 10X buffer || 1.5
| 10 uM fwd primer || 1.0
|-
|-
| EcoRI || 1.0
| 10 uM rev primer || 1.0
|-
|-
| PstI || 1.0
| 2x GoTaq green || 12.5
|-
|-
| dH<sub>2</sub>O || 9.5
| dH<sub>2</sub>O || 10.3
|-
|-
| &nbsp; || 15 μL --> 37°C/ ~15 min.
| &nbsp; || 25.0
|}
|}
Program: GOTAQ35cyc
* 95°C, 3 min
* 30x[95°C, 1 min; 57°C, 1 min; 72°C, 3 min]
* 72°C, 3 min
* 4°C ∞


----
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Revision as of 10:48, 27 January 2015

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01/27/15

  • Gel purification - KAH60 vector PCR, ATF2 PCR
  • PCR - re-amplify KAH 60 vector


Gel purification

  • Gel purify fragments from PCR done on 01/22/15
  • KAH60/pcVN - 6637 bp
  • ATF2 - 960 bp

PCR

  • Use purified KAH60 fragments as template for PCR
  1. KAH60 "backbone" (without VP64, includes scars) = 6637: KAH60 F1/ KAH60 R1


Reagent Rxn1 Expected:
1. KAH60 = 6637 		Hover name
10 μL/lane, 1% agarose; Ladder
Template 0.2
10 uM fwd primer 1.0
10 uM rev primer 1.0
2x GoTaq green 12.5
dH2O 10.3
  25.0

Program: GOTAQ35cyc

  • 95°C, 3 min
  • 30x[95°C, 1 min; 57°C, 1 min; 72°C, 3 min]
  • 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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