User:Daniel A. Vargas/Notebook/General lab notebook/2015/08/04

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ASSEMBLY of MV10, ATF2, and Gal4DB/mCherry

  • LCR Assembly



LCR Assembly

Prep the Bridge Oligos (BO)

  1. MV10/Gal4DB-mCh bridge: LCRb_MV10tctGal4DB_rc
  2. Gal4DB-mCh/ATF2 bridge: LCRb_mCh_ATF2_rc
  3. ATF2/MV10 bridge: LCRb_ATF2tcaMV10_rc
  • Overview - Bridge Oligos need to be brought to a working concentration of 300 nanomolar (nM). Eventually, final conc. in LCR rxn. will end up being 30 nanomolar (nM) each.

Table: Make a 300 nM working solution (final volume = 100 μL) in a fresh tube for each Bridge Oligo (BO) (in 1.5 mL tubes)

Reagent BO 1 BO 2 BO 3
100 μM Stock BO 3.0 3.0 3.0
dH2O 97.0 97.0 97.0
  100.0 100.0 100.0


Prep the dsDNA fragments

  1. MV10 (XbaI-cut and mung bean-blunted)
  2. Gal4DB-mCherry (Phusion PCR)
  3. ATF2 (Phusion PCR)
  • Overview - dsDNA will be diluted to a concentration of 30 fmol/μL
  • Calculations: The volume of purified dsDNA (x) you will need to dilute in a final volume of 50 μL = length in bp ÷ measured ng/μL * 30 fmol/μL * 650 fg/fmol dsDNA ÷ 1,000,000 fg/ng * 50 μL final volume.
    • MV10: length in bp ÷ measured ng/μL * 0.0195 ng/μL * 50μL = x μL
    • Gal4DB-mCherry: length in bp ÷ measured ng/μL * 0.0195 ng/μL * 50μL = x μL
    • ATF2: length in bp ÷ measured ng/μL * 0.0195 ng/μL * 50μL = x μL


Table: Dilute each purified dsDNA to 30 fmol/μL (30 nM) in a final volume of 50 μL (in 1.5 mL tubes)

Reagent DNA 1 DNA 2 DNA 3
DNA x μL x μL x μL
dH2O ## ## ##
  50.0 50.0 50.0


PNK treatments

  1. MV10 + Gal4DB-mCh + ATF2
  2. MV10 (neg ctrl.)

Overview: Mix double-stranded DNA fragments, treat with PNK to add phosphate groups

  • Dilute the purified dsDNA to 30 fmol/μL (30 nM) in a final volume of 50 μL
  • Calculation: The volume of purified dsDNA (x) you will need to dilute in a final volume of 50 μL = length in bp ÷ measured ng/μL * 30 fmol/μL * 650 fg/fmol dsDNA ÷ 1,000,000 fg/ng * 50 μL final volume.
    Formula: x μL = length in bp ÷ measured ng/μL * 0.0195 ng/μL * 50μL

Table: Set up the following in PCR tubes:

Reagent PNK 1 PNK 2
30 fmol/μL MV10 2.0 2.0
30 fmol/μL Gal4DB-mCh 2.0 ---
30 fmol/μL ATF2 2.0 ---
10x T4 Ligation buf (NEB) 1.0 1.0
T4 PNK (NEB) 0.5 0.5
dH2O 2.5 6.5
  10.0 μL 10.0 μL
  • Thermal cycler: program the following...
    • Incubate at 37°C/ 30 min.
    • Heat-inactivate PNK at 65°C/ 20 min.


LCR Reactions

  1. MV10 + Gal4DB-mCh + ATF2
  2. MV10 (neg. ctrl.)
  • Using the entire PNK DNA reaction, add other components (into the same PCR tubes) as described in the table below
  • Best way to do this is to make a master mix, then aliquot into PNK DNA
  • Master mix multiplier = number of LCR reactions + 1 (for pipetting error)

Master Mix (in 1.5 mL tube)

Reagent (Single Rxn.) MM x3
Oligo Bridge 1 (2.0) 6.0
Oligo Bridge 2 (2.0) 6.0
Oligo Bridge 3 (2.0) 6.0
10X Ampligase Buffer (2.0) 6.0
Ampligase (1.0) 1.0
dH2O (1.0) 3.0
  (10.0) 30.0

Table: Add master mix directly into 10 μL PNK DNA tubes

Reagent LCR 1 LCR 2
PNK DNA 10.0 10.0
Master Mix 10.0 10.0
  20.0 20.0
  • Run LCR...
    • Look for "LCR" program on one of the PCR machines

Program: LCR

  • 94°C, 2 min
  • 50x[94°C, 10 sec; 55°C, 30 sec; 66°C, 60 sec]
  • 4°C ∞


Transformation



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