Example:
Amplifying the backbone plasmid sequence and the Super Folder GFP by PCR
You will need:
1. A Thermal Cycler
2. Thin walled PCR tubes (e.g. Axygen cat no. EPE013+ 0.2ml PCR thin walled tubes) 3. 10 μM forward and reverse primer pairs (from Biolegio, Invitrogen or Sigma)
4. 10 mM dNTPs (e.g. NEB cat no. N-0446S dNTP set 4x 100 mM 25 μmol) 5. PCR grade Nuclease free water (e.g. Invitrogen cat no. AM9938 Nuclease free water) 6. Phusion High Fidelity DNA polymerase and HF buffer (e.g. NEB cat no. F-530S) 7. Template DNA (10 pg-1 ng)
The PCR and cycling conditions are as follows using the reaction conditions recommended by the manufacturer for using Phusion High fidelity DNA polymerase (www.finnzymes.com):
Two PCR reactions are set up to generate the plasmid backbone (without RFP) and the super folder GFP. The template DNAs will be pSB3K3 BBa_J69512 (containing the mCherry Biobrick: BBa_J06504) and pSB3K3 BBa_I746914 (containing the super folder GFP Biobrick: BBa_I746919).
You will need:
1. A Thermal Cycler
2. Thin walled PCR tubes (e.g. Axygen cat no. EPE013+ 0.2ml PCR thin walled tubes) 3. 10 μM forward and reverse primer pairs (from Biolegio, Invitrogen or Sigma)
4. 10 mM dNTPs (e.g. NEB cat no. N-0446S dNTP set 4x 100 mM 25 μmol) 5. PCR grade Nuclease free water (e.g. Invitrogen cat no. AM9938 Nuclease free water) 6. Phusion High Fidelity DNA polymerase and HF buffer (e.g. NEB cat no. F-530S) 7. Template DNA (10 pg-1 ng)
The PCR and cycling conditions are as follows using the reaction conditions recommended by the manufacturer for using Phusion High fidelity DNA polymerase (www.finnzymes.com):
Two PCR reactions are set up to generate the plasmid backbone (without RFP) and the super folder GFP. The template DNAs will be pSB3K3 BBa_J69512 (containing the mCherry Biobrick: BBa_J06504) and pSB3K3 BBa_I746914 (containing the super folder GFP Biobrick: BBa_I746919).
PCR
Materials
Color Label Contents
Yellow 'F' 25 mM GFP primer forward
Yellow 'R' 25 mM GFP primer reverse
Green 'G' GFP template
White 'F' 25 mM vector primer forward
White 'R' 25 mM vector primer reverse
Black 'V' vector template
Red 'dNTP' 10 mM dNTPs
Blue 'B' 5x HF Phusion buffer
Protocol:
Perform all steps on ice!
Make up two reactions (one with GFP primers/template, one with vector primers/template).
Reaction contents:
H2O 35.5 ul
5x HF Phusion Buffer 10 ul
10 mM dNTPs 1 ul
25 mM forward primer 1 ul
25 mM reverse primer 1 ul
template DNA 1 ul
Phusion polymerase 0.5 ul
Total 50 ul
Add the components in the order listed.
Materials
Color Label Contents
Yellow 'F' 25 mM GFP primer forward
Yellow 'R' 25 mM GFP primer reverse
Green 'G' GFP template
White 'F' 25 mM vector primer forward
White 'R' 25 mM vector primer reverse
Black 'V' vector template
Red 'dNTP' 10 mM dNTPs
Blue 'B' 5x HF Phusion buffer
Protocol:
Perform all steps on ice!
Make up two reactions (one with GFP primers/template, one with vector primers/template).
Reaction contents:
H2O 35.5 ul
5x HF Phusion Buffer 10 ul
10 mM dNTPs 1 ul
25 mM forward primer 1 ul
25 mM reverse primer 1 ul
template DNA 1 ul
Phusion polymerase 0.5 ul
Total 50 ul
Add the components in the order listed.