User:Karmella Haynes/Notebook/BioBrick cloning/2015/05/18: Difference between revisions

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* Phusion PCR
* Phusion PCR
# DBN006 / DBN003 f1 (Tm 71.6), DBN002 r1 (Tm 59.4)
# DBN006 / DBN003 f1 (Tm 71.6), DBN002 r1 (Tm 59.4)
# same


{| {{table}} border="1" cellspacing="3" <!-- Phusion PCR table -->
{| {{table}} border="1" cellspacing="3" <!-- Phusion PCR table -->
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* 4°C ∞
* 4°C ∞
Conclusion
Conclusion
* tba
* Failed - Tm too high. The binding site for one of the primers is actually 40°C. Try again tomorrow with GoTaq & lower annealing temp.




Revision as of 17:06, 18 May 2015

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05/18/15

  • Ben - assembly: HPK-CFP into homology arms
  • Cas-tone - modification #1


Ben - assembly: HPK-CFP into homology arms

  • Phusion PCR
  1. DBN006 / DBN003 f1 (Tm 71.6), DBN002 r1 (Tm 59.4)
  2. same
Reagent Volume Mix (x2) Expected:
1,2. DBN006 = 1972 		Hover name
5 μL/lane; 1% agarose; Ladder
DNA(plasmid) 0.5 μL 1.0
10 μM F primer 1.0 2.0
10 μM R primer 1.0 2.0
10 mM dNTPs 1.0 2.0
Phusion Pol. 0.5 1.0
5X HF buffer 10.0 20.0
dH2O 36.0 72.0
  50.0

Program: Phusion (block B)

  • 98°C, 3 min
  • 35x[98°C, 10 sec; 57°C, 30 sec; 72°C, 30 sec]
  • 72°C, 10 min
  • 4°C ∞

Conclusion

  • Failed - Tm too high. The binding site for one of the primers is actually 40°C. Try again tomorrow with GoTaq & lower annealing temp.


  • PCR clip & clone
    • Qiagen column clean-up



Cas-tone Project

  • STAGE 1 - pcDNA-dCas9-VP64 vector re-design
    • Knock-out VP64 from C-terminus - replace AscI-VP64:HA:STOP-EcoRI-XbaI with AscI-HA:STOP-XbaI dsOligo
    • DONE - Knock-out FLAG from N-terminus - replace SpeI-CMV:3xFLAG:NLS-SacII with PCR-amplified (SpeI)/XbaI-CMV-SpeI-NotI-SacII; use reverse primer to add SacII
  • STAGE 2 - Histone parts
    • PCR-clone histone parts as XbaI-Kozak-histone part-a-NotI. Note: Do no use Kozak BioBrick, since this will introduce unnatural amino acids onto conserved histone N-terminal tail
    • DONE - Order primers (annotated in Benchling)
  • STAGE 3 - Cas-fusion
    • Insert Kozak-histone parts (X/N) into dCas9 (S/N)
  • STAGE 4 - gRNA
    • Put pre-existing gRNA (from luc experiment) into pSPgRNA


Assembly

  1. CMV-dCas-HA: Cas47107_Mod1/(dsOligo)/50 + CMV-dCas-VP64/(AscI/XbaI)/9453


  • Digests (Fermentas FD)
    • Specific notes
Reagent Volume
DNA (plasmid) 15.0
10x buffer 3.0
AscI 1.0
XbaI 1.0
dH2O 10.0
  30 μL

--> 37°C/ ~30 min.

  • Gel purify
    • Sigma GenElute kit; elute & back-elute w/ 25 μL elution sln.


  • Measure conc.'s
Sample OD260 260/280 ng/μL
1. CMV-dCas-VP64/(AscI/XbaI) --- --- ---


  • Phosphorylate and anneal oligo pair
  1. Cas47107_Mod1 top/btm
Reagent Rxn Mix (2x)
100 μM Oligo 1 1.0 2.0
100 μM Oligo 2 1.0 2.0
10x T4 Lign buf (NEB) 1.0 2.0
T4 PNK (NEB) 0.5 1.0
dH2O 6.5 13.0
  10.0

LabNet OptiMax Thermocycler: AnOlig RD

  • 37°C, 30 min
  • 95°C, 5 min
  • Ramp down to 25°C, 5°C/1 min. [90/1 min, 85/1 min, 80/1 min, ... 25°C/1 min]
  • 25°C, ∞

Dilute the product(s) 1:250

  • Add 2 μL product to 498 μL dH2O


  • Ligations
    • 50 bp insert / 9453 bp vector * 2 * 100 ng vector = 1.06 ng insert
  1. Cas47107_Mod1(1:250 dsOligo) + CMV-dCas-VP64(AscI/XbaI)
  2. CMV-dCas-VP64(AscI/XbaI)
  1 2
Insert DNA 2.0 ---
Vector DNA ### ###
2x lgn buf (Roche) ### ###
T4 ligase (NEB) 1.0 1.0
dH2O ### ###
  # μL # μL



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