Step Description Date/Initials Link protocol Link results
1 Digest pDN0501 / pDN0502 with HidIII-HF and NotI-HF 20191118KM
2 Amplify DHFR-attL1 fragments ordered by TWIST 20191122DEY
3 Digest amplified DHFR-attL1 fragments with HidIII-HF and NotI-HF
4 T4 ligation
5 Transformation to ccdB survival cells

Material

Check Reagent Supplier Cat. no Primer/Sample ID Memo
Nuclease free ddH2O Invitrogen 10977-015
Phusion HF buffer (5x) NEB B0518S
25mM dNTPs TaKaRa
Frd Primer FASMAC POEM3 TWIST Frd
Rvs Primer FASMAC POEM4 TWIST Rvs
Phusion pol - KM2 Home brew Phusion pol
Template DNA TWIST - POEM1, POEM2 Just over 300 bp
Agarose RIKAKEN RSV-AGRP-500G
1x TBE buffer - - Homebrew (link:)
ExcelBand 100 bp DNA Ladder SMOBiO DM2100
5x Loading dye - - Homebrew (link:)
Midori green Advance Nippon Genetics NE-MG04

Before preparing: Check your primer Tm at NEB website (http://tmcalculator.neb.com/#!/main with Phusion/HF buffer setting)






Protocol

1 Reaction preparation
  • Mix the following reagents in one tube (ideally from top to bottom)
Check Reagent Volume (µl) 1 reaction Volume (µl) _ _ _ reaction Final concenration Memo
ddH20 6.62 -
Phusion HF buffer (5x) 2.0 -
25mM dNTPs 0.08 0.2 mM Equal volume mix of 100mM dA/T/G/CTPs will give this solution.
Frd Primer 0.05 0.5 µM
Rvs Primer 0.05 0.5 µM
Transfer the tube on ice
Phusion pol 0.2 - Home brew Phusion pol
Template 1 - 1ng is enough for template.
Total (µL) 10









2 Reaction
  • Run the following program on a thermal cycler
STEP Action Time Memo
1 98˚C 30 sec Initial denaturation.
2 98˚C 10 sec Denaturation.
3 63-68˚C 10 sec Annealing. Expected Tm:68˚C. Gradient for optimization.
4 72˚C 15 sec Extension. 15sec/kbp, 300bp < 1kbp
5 GOTO 2 x 34
6 72˚C 3:00 Final extension.
7 4˚C FOREVER Stop reaction.



3 Agarose gel electrophioresis
  • Make agarose gel
Reagent Amount Memo
Agarose powder 2.0 g 2.0%(w/v)
1x TBE buffer 100 mL
Microwave and dissolve agarose completely.
Midori green advance 10 µL DNA stain
Swirl to mix the stain uniformly, and cast gels. 100 mL will give you 2 x wide + 2 x small on heat-tolerant trays.
Wait for gel to settle.





Dan Yamamoto-Evans 2019/11/22 14:47

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  • interactome/experiments/20191122.txt
  • Last modified: 2019/11/22 14:48
  • by dan