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Name:
Date:
Project:
gene of interest:
vector of interest:
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 primer | 2.5µL | |
| 10µM reverse primer | 2.5µL | |
| 100% DMSO | 0 - 6µL | |
| 5x HF/GC Buffer | 10µL | |
| dNTPs mix, 10mM each | 1µL | |
| ddH2O | ++50µL | |
| Phusion-HF polymerase | 1µL | |
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 | Time | Cycles |
|---|---|---|
| 98°C | 30 seconds | 1 |
| 98°C | 10 seconds | 18-30 |
| ____ °C* | ____ seconds* | |
| 72°C | 30 seconds/1kB | |
| 72°C | 10 minutes | 1 |
| 10°C | hold | 1 |
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
- continue with ligation
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