User:Karmella Haynes/Notebook/BioBrick cloning/2015/06/22: Difference between revisions

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(Autocreate 2015/06/22 Entry for User:Karmella_Haynes/Notebook/BioBrick_cloning)
 
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==mm/dd/yy==
==mm/dd/yy==
<!-- Precede finished items with a checkmark &#x2713; -->
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* Line item 1
* Rene - CRISPR PCR library PCR
* Line item 2
* Line item 2
----
'''Rene - CRISPR PCR library PCR'''
* '''Part 1 - GoTaq "Dirty" PCR on liquid mini-cultures, 600 bp amplicons; primers P163/P215'''
* Part 2 - Nested SYBR real time PCR & melt curves on (1) mini cultures, (2) amplicons from GoTaq
* Part 1 - GoTaq "Dirty" PCR on liquid mini-cultures, 600 bp amplicons
** Clone labeling notes:
*** Lu34 = Luc14 cells treated with gRNA034
*** Ga34 = Gal4EED/luc cells treated with gRNA034
*** A/B = 96-well plate A or B
*** A01, A02, etc. = well position in dish or spot on agar array
1-96. Luc14 g034 - Lu34_AA01 - Lu34_AH12 (full plate)<br>
{| {{table}} cellspacing="3" <!-- PCR rxn. table -->
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Rxn1-24
| bgcolor=#cfcfcf | Mix (x104)
| rowspan="7" | Expected:<br>1-14. PCR insert/ pJET = ~600 bp
| rowspan="7" | [[Image:KAH052615_gel3.jpg|250px|Hover name]]<br>10 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
|-
| Template (culture) || 2.0 || ---
|-
| 10 uM fwd primer || 1.0 || 104.0
|-
| 10 uM rev primer || 1.0 || 104.0
|-
| 2x GoTaq (clear) || 12.5 || 1300.0
|-
| dH<sub>2</sub>O || 8.5 || 884.0
|-
| &nbsp; || 25.0 μL
|}
* Note: Master Mix Multiplier = 96 + 1 extra 8-well column = 104
* Aliquot 200 μL into 8 tubes in 8-tube strip
* Use multichannel to aliquot 23 μL to 96-well plate, 1 column at a time
* Refill the 8-tube strip as needed with enough to fill remaining columns
PCR - Labnet: "GoTaq35KAH"
* 95°C, 3 min
* 35x[95°C, 10 sec; 60°C, 10 sec; 72°C, 20 sec]
* 72°C, 3 min
* 4°C ∞
CONCLUSIONS
* tba




Revision as of 13:58, 22 June 2015

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mm/dd/yy

  • Rene - CRISPR PCR library PCR
  • Line item 2


Rene - CRISPR PCR library PCR

  • Part 1 - GoTaq "Dirty" PCR on liquid mini-cultures, 600 bp amplicons; primers P163/P215
  • Part 2 - Nested SYBR real time PCR & melt curves on (1) mini cultures, (2) amplicons from GoTaq


  • Part 1 - GoTaq "Dirty" PCR on liquid mini-cultures, 600 bp amplicons
    • Clone labeling notes:
      • Lu34 = Luc14 cells treated with gRNA034
      • Ga34 = Gal4EED/luc cells treated with gRNA034
      • A/B = 96-well plate A or B
      • A01, A02, etc. = well position in dish or spot on agar array

1-96. Luc14 g034 - Lu34_AA01 - Lu34_AH12 (full plate)


Reagent Rxn1-24 Mix (x104) Expected:
1-14. PCR insert/ pJET = ~600 bp 		Hover name
10 μL/lane, 1% agarose; Ladder
Template (culture) 2.0 ---
10 uM fwd primer 1.0 104.0
10 uM rev primer 1.0 104.0
2x GoTaq (clear) 12.5 1300.0
dH2O 8.5 884.0
  25.0 μL
  • Note: Master Mix Multiplier = 96 + 1 extra 8-well column = 104
  • Aliquot 200 μL into 8 tubes in 8-tube strip
  • Use multichannel to aliquot 23 μL to 96-well plate, 1 column at a time
  • Refill the 8-tube strip as needed with enough to fill remaining columns


PCR - Labnet: "GoTaq35KAH"

  • 95°C, 3 min
  • 35x[95°C, 10 sec; 60°C, 10 sec; 72°C, 20 sec]
  • 72°C, 3 min
  • 4°C ∞


CONCLUSIONS

  • tba



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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