POEM project | N-term DHFR-PCA plasmid construction

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Overview

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 20191123DEY
4 T4 ligation
5 Transformation to ccdB survival cells

Material

Check Reagent Supplier Cat. no Primer/Sample ID Memo
Purified PCR product POEM5 & POEM6
ddH2O Invurtogen 10977-015
HindIII-HF NEB R3104S 37C, Inactivated at 80C
NotI-HF NEB R3189S 37C, Inactivated at 65C
10x Cutsmart buffer NEB B7204
FasGene PCR clean kit Nippon Genetics FG-91302








Protocol

1 Reaction preparation
  • Mix the following reagents in one tube (ideally from top to bottom)
Check Reagent Volume (µl) per reaction Final concenration Memo
ddH20 34 -
10x CS buffer 5 1 x
DNA 10 1 µg should be enough
NotI-HF 1 20U/µL
HindII-HF 1 20U/µL
Total (µL) 50



2 Reaction
  • Run the following program on a thermal cycler
STEP Action Time Memo
1 37˚C 16 hours Reaction
2 80˚C 20 mins Inativation
7 4˚C FOREVER Stop reaction.



3 Cleanup purification
  • Purify product using FastGene PCR clean kit.
  • Measure concentration





— Dan Yamamoto-Evans 2019/11/23 12:15

Print at 96%

  • interactome/experiments/20191123.txt
  • Last modified: 2019/11/23 12:15
  • by dan