User:Karmella Haynes/Notebook/BioBrick cloning/2015/05/18

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Karmella's BioBrick Cloning Main project page
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05/18/15

  • Ben - assembly: HPK-CFP into homology arms
  • Cas-tone - modification #1
  • Rene - PCR cloning trial 3



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 49°C. Redesign this primer.



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
    • DONE - Order primers (annotated in Benchling)
    • 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
  • 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 Hover name
30 μL/lane, 1% agarose; Ladder
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) 0.041 2.005 40.5
1. CMV-dCas-VP64 miniprep 0.19 1.93 189.5
1. Luc14 g034 0.035 1.97 34.8
1. Gal4EED g034 0.039 1.867 38.7


  • 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)
Reagent Rxn1 Rxn2
Insert DNA 2.0 ---
Vector DNA 1.5 1.5
2x lgn buf (Roche) 5.0 5.0
T4 ligase (NEB) 1.0 1.0
dH2O 0.5 2.5
  10.0 μL 10.0 μL


RESULTS (5/19/15)

  • Success! ~100 colonies on ligation plate, 6 on neg ctrl.
  • Pick two colonies for 5mL cultures



Rene - PCR cloning trial 3

  • This time use Roche ligation buffer and NEB T4 ligase
  • Use the CloneJET PCR Cloning Kit - MAN0012707 CloneJET PCR Cloning (manual)
    • pJET1.2/blunt vector is 5'-phosphorylated and accepts blunt products (Phusion PCR)
    • All common laboratory E. coli strains can be directly transformed with the ligation product
    • Re-circularized vector expresses toxic enzyme, no blue/white screening needed
    • Optimal insert : vector (0.05 pmol ends) ratio is 3:1; Calculation: length of insert DNA x 0.05 = ng insert to use


  • Ligations & transformations - CloneJET PCR Cloning Kit
  1. Luc14 + CRISPR g034; size = 630 (use 31.5 ng)
  2. Gal4EED/luc + CRISPR g034; size = 630 (use 31.5 ng)


Reagent Volume
2x Roche lig buf 5.0
PCR product (>31.5 ng) 1.0
pJET1.2/blunt vector 1.0
NEB T4 ligase 1.0
dH2O 2.0
  10.0 μL
  • Incubate the ligation mixture at room temperature (22°C) for 5 min.
  • Transform 50 μL DH5α-turbo with 10 μL ligation reaction. Follow short protocol.


RESULTS (5/19/15)

  • Success! ~200 colonies on each plate. Pick for 96-well "mass culture" plates



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