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Name:

Date:

Project:


gene of interest:
vector of interest:


PrimerNameSequence 5' - 3'Tm/°CTm binding/°C
1



2



3



4



Restriction EnzymeName
1
2
3
4



Outline


  • PCR to amplify inserts for subsequent cloning using Phusion-HF polymerase
  • purification kit (when using PCR product as insert)
  • restriction digest insert
  • restriction digest vector
  • CIP treatment of vector
  • preparative agarose gel
  • purification of DNA from agarose using commercial kit like “Qiaquick gel purification kit”
  • determine concentration (nanodrop)
  • ligation
  • 5-10µL of ligation mix: Transformation in E.coli Top10,XL10-gold or DB3.1
  • DNA miniprep
  • sequencing


To Do


Please ensure, the following is at hand and prepared in sufficient amounts:

  • DNA mini/midi prep of your template vector of interest
  • DNA mini/midi prep of your template insert of interest
  • kits and all components


  • well designed primers
  • Agarose Gel components such as agarose, TAE or TBE buffer, DNA loading dye and DNA gel dye (GelRed)
  • restriction enzymes and fitting buffers
















Reaction Setup



plasmid DNA as template



plasmid DNA 20ng/µL
_____µL
10µM forward primer2.5µL
10µM reverse primer2.5µL
100% DMSO0 - 6µL
5x HF/GC Buffer
10µL
dNTPs mix, 10mM each1µL
ddH2O++50µL
Phusion-HF polymerase1µL

table 02 - PCR reaction for plasmid DNA


  • standard usage of template is 20ng, can be varied bewteen 10 and 50ng if needed
  • dilute 100µM Primer stocks 1:10 for 10µM working solution
  • calculate the amount of water needed and put at first
  • then add the Buffer and mix well
    • 5x HF Buffer is standard, use 5x GC Buffer if the gene of interest shows a GC content higher than 60%
  • DMSO can be added to facilitate the annealing of primers to the template, enhancing the amplification
  • add all other components to a final volume of 50µL
  • mix everything well
  • spin down PCR tubes to ensure no drops are sticking to the tube wall
  • add Phusion-HF polymerase at last


NOTE

Please prepare the PCR mix on ice. Buffers can be thawed at room temperature.



PCR program


Temperature
TimeCycles
98°C30 seconds1
98°C10 seconds18-30
____ °C*____ seconds*
72°C30 seconds/1kB
72°C10 minutes1
10°Chold1

table 03 - PCR program

  • initial heating step of 98°C for 30seconds required to activate Phusion-HF polymerase, also works as an inital denaturating step of plasmid DNA, can be increased up tp 180sec
  • further cycles can be altered in: number of cycles, annealing temperature and time, elongation time
  • run the PCR products on an Agarose Gel
  • cut the gene of interest from the gel and purify using NucleoSpin Gel and PCR Clean-up KIT (Macherey-Nagel, 740609.250)
  • determine DNA concentration using NanoDrop in SPC lab, with Kit- Elution Buffer as a blank





*primer annealing is primer depended, for temperature please refer to the data sheet provided by your primer supplyer, time standards are 10 to 60 seconds








Restriction Digest


Insert restriction digest

PCR product / plasmid DNA
_______µL

enzyme 1: __________, 10U/µL (NEB)*
1µL

enzyme 2: __________, 10U/µL (NEB)*1µL

10x CutSmart Buffer (NEB)5µL

ddH2O++50µL


  • calculate the amount of water needed and place first in 1.5mL Eppi
  • add the 10x CutSmart Buffer and mix well
  • add your DNA
  • add the restriction enzymes at last
  • mix well by tipping the Eppi or mix with pipetting up and down
  • 1µg of PCR product are standard amounts to digest
  • 5–10 units of enzyme per µg DNA are recommended by NEB
  • reaction equals 50µL
  • incubate at 37°C for 1h or

NOTE

Please prepare the digestion mix on ice.

Vector restriction digest

vector DNA (mini or midi)
______µL

enzyme 1: __________, 10U/µL (NEB)*1µL

enzyme 2: __________, 10U/µL (NEB)*1µL

10x CutSmart Buffer (NEB)**5µL

ddH2O++50µL

  • calculate the amount of water needed and place first in 1.5mL Eppi
  • add the 10x CutSmart Buffer and mix well
  • add the vector DNA
  • add the restriction enzymes at last
  • mix well by tipping the Eppi or mix with pipetting up and down
  • 1-2µg of vector DNA are standard amount to digest
  • keep in mind to adjust the amount of enzyme, when you use more than 1µg
  • 5-10 units of enzyme per µg of DNA are recommended by NEB
  • reaction equals 50µL
  • incubate at 37°C for 1h


* for our cataloge of available restriction enzymes please refer to our NEB freezer






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