Step Description Date/Initials Link protocol Link results
1 Total RNA extraction from HEK293T cells 20191122DEY
2 1st strand cDNA synthesis 20191122DEY
3 Target gene amplification from cDNA sample 20191123DEY
4 BP-reaction
5 Sanger sequence check

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 PI11 to PI20
Rvs Primer FASMAC PI11 to PI20
Phusion pol - KM2 Home brew Phusion pol
Template cDNA - Prepared on 20191122. Sample1
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

List of paramater tested.

RT + - + - + - + - + -
Tm/Target gene MORF4L1 MORF4L1 MRFAP1 MRFAP1 BCL2L2 BCL2L2 BIK BIK MRFAP1L1 MRFAP1L1
Row/Col on PCR plate 1 2 3 4 5 6 7 8 9 10
69.0 A
B
67.9 C
D
65.4 E
F
63.6 G
H





  • Mix the following reagents in one tube (ideally from top to bottom)
Check Reagent Volume (µl) 1 reaction Volume (µl) for 25 reactions with 20µL vol Final concenration Memo
ddH20 6.62 331 -
Phusion HF buffer (5x) 2.0 100 -
25mM dNTPs 0.08 4 0.2 mM Equal volume mix of 100mM dA/T/G/CTPs will give this solution.
cDNA template 1 50 Diluted cDNA sample.
Transfer the tube on ice
Phusion pol 0.2 10 - Home brew Phusion pol
Aliquot 18 µL to each tube
10µM Frd Primer 0.5 1 0.5 µM
10µM Rvs Primer 0.5 1 0.5 µM
Total (µL) 10 20



2 Reaction
  • Run the following program on a thermal cycler
STEP Action Time Memo
1 98˚C 10 sec Initial denaturation.
2 98˚C 10 sec Denaturation.
3 63-68˚C 10 sec Annealing.
4 72˚C 1 min Extension. 15sec/kbp, but using longer
5 GOTO 2 x 34
6 72˚C 10:00 Final extension.
7 4˚C FOREVER Stop reaction.



3 Agarose gel electrophioresis
  • Make agarose gel
Reagent Amount Memo
Agarose powder 1.0 g 1.0%(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/20191123-2.txt
  • Last modified: 2019/11/23 13:38
  • by dan