User:Karmella Haynes/Notebook/BioBrick cloning/2015/04/21: Difference between revisions

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* Assemblies
* Assemblies
# AubR/MRV: AubRPCR(E/X)/851 + MRV(E/X)/size
# AubR/MRV: AubR-PCR(E/X)/851 + MRV(E/X)/3201
# BjaR/MRV: BjaRPCR(E/X)/776 + MRV(E/X)/size
# BjaR/MRV: BjaR-PCR(E/X)/776 + MRV(E/X)/3201
# BraR/MRV: BraRPCR(E/X)/776 + MRV(E/X)/size
# BraR/MRV: BraR-PCR(E/X)/776 + MRV(E/X)/3201
# RpaR/MRV: RpaRPCR(E/X)/779 + MRV(E/X)/size
# RpaR/MRV: RpaR-PCR(E/X)/779 + MRV(E/X)/3201




Line 74: Line 74:
| Sample || OD260 || 260/280 || ng/μL
| Sample || OD260 || 260/280 || ng/μL
|-
|-
| 1. Digested part (a/b) || --- || --- || ---
| 1. AubR-PCR (E/X) || -0.005 || 5.875 || -5.001
|-
|-
| 2. Digested part (c/d) || --- || --- || ---
| 2. BjaR-PCR (E/X) || -0.005 || 9 || -4.788
|-
| 3. BraR-PCR (E/X) || -0.005 || 7.143 || -5.291
|-
| 4. RpaR-PCR (E/X) || -0.003 || 29 || -3.052
|-
| 5. MRV (E/X) || 0.007 || 1.388 || 7.11
|}
|}


* Note: unsure why concentrations so low. Proceed with ligations anyway, see what happens.


* Ligations
* Ligations

Revision as of 15:50, 21 April 2015

Karmella's BioBrick Cloning <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
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04/21/15

  • Ryan - regulator assemblies
  • Rene - check gRNA assemblies; digests & sequencing
  • LCR Development - Phusion PCR


Ryan - Receiver plasmids, assembly

  • Stage 1 - insert Regulator ORFS
    • DONE - Phusion PCR-amplify ORFs (add 5'-E, 3'-X cut sites)
    • Insert ORF(E/X) into Vector (E/X). see Vector plasmid map
  • Stage 2 - insert Promoters
    • Cut & dephos promoters with E/X
    • Insert promoters(E/X) into Vector/Regulator (BbsI) via Lig/Dig cycling


  • Clean up PCR products
    • Zymo clean and concentrator
    • Elute w/ 25 μL dH2O


  • Assemblies
  1. AubR/MRV: AubR-PCR(E/X)/851 + MRV(E/X)/3201
  2. BjaR/MRV: BjaR-PCR(E/X)/776 + MRV(E/X)/3201
  3. BraR/MRV: BraR-PCR(E/X)/776 + MRV(E/X)/3201
  4. RpaR/MRV: RpaR-PCR(E/X)/779 + MRV(E/X)/3201


  • Digests
    • E/X
  1. AubR, 851 bp
  2. BjaR, 776 bp
  3. BraR, 776 bp
  4. RpaR, 779 bp
  5. Modular Receiver Vector (MRV)
Reagent Rxn 1-4 Rxn 5
DNA 10.0 5.0
10X buffer 3.0 3.0
EcoRI 1.0 1.0
XbaI 1.0 1.0
dH2O 15.0 20.0
  30.0 30.0


  • Column clean-up
    • Zymo clean and concentrator


  • Measure conc.'s
Sample OD260 260/280 ng/μL
1. AubR-PCR (E/X) -0.005 5.875 -5.001
2. BjaR-PCR (E/X) -0.005 9 -4.788
3. BraR-PCR (E/X) -0.005 7.143 -5.291
4. RpaR-PCR (E/X) -0.003 29 -3.052
5. MRV (E/X) 0.007 1.388 7.11
  • Note: unsure why concentrations so low. Proceed with ligations anyway, see what happens.
  • 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


Rene - check gRNA assemblies

  • Minipreps from 4/20/15
    • Note: ran out of time last week to grow up cultures. Stored at 4C until this week, grew for ~5 hours and then miniprepped w/ Sigma GenElute kit (elution vol. = 75 μL)
  • Check w/ BpiI/XbaI
  1. gRNA51, 1
  2. gRNA51, 2
  3. gRNA52, 1
  4. gRNA52, 2
  5. gRNA53, 1
  6. gRNA53, 2 (Note: Accidentally mixed 6 w/ 7 during pellet resuspension!)
  7. gRNA54 (Note: Accidentally mixed 6 w/ 7 during pellet resuspension!)
  8. gRNA55, 1
  9. gRNA55, 2
  10. gRNA56, 1
  11. gRNA56, 2
  12. pX330 (ctrl)


Reagent Rxn 1-11 Mix(x13) Expected
pX330 = ~8335, 195
gRNA+pX330 = 8530 		Hover label
DNA 3.0 ---
10X buffer 1.5 19.5
BpiI 0.5 6.5
XbaI 0.5 6.5
dH2O 9.5 123.5
  15.0



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