O
P
Phosphate Buffered Saline 10X (PBS)
1 L 4 L
NaCL 80 g 320 g
KCL 2 g 8 g
Na2HPO4 14.4 g 57.6
or
Na2HPO4-7H20 26.8 g 107.2 g
KH2PO4 2.4 g 9.6 g
pH to 7.4 with 10 M NaOH and QS to volume
P
Phosphate Buffered Saline 10X (PBS)
1 L 4 L
NaCL 80 g 320 g
KCL 2 g 8 g
Na2HPO4 14.4 g 57.6
or
Na2HPO4-7H20 26.8 g 107.2 g
KH2PO4 2.4 g 9.6 g
pH to 7.4 with 10 M NaOH and QS to volume
PBS EDTA
For 2 L:
40 ml 0.5 M EDTA
200 ml 10X PBS
QS to 2 L.
Proteinase K Reaction Buffer
0.01 M Tris, pH 7.8
0.05 M EDTA
0.5% SDS
Proteinase K Digestion (Example using Borrelia burgdorferi, for the removal of
surface proteins)
950 µl Borrelia at 1 x 10-9 (1000,000,000) + 50 µl Proteinase K (4 mg/ml stock)
E/E RT 10 min
Stop reaction with 10 µl PMSF (0.1 M)
Potassium Chloride (0.5 M)
9.32 g QS to 250 ml with ddH2O
PCR (example)
73.5 µl H2O
10 µl 10X buffer (supplied with the polymerase)
2 µl DNA (this amount/concentration highly affects outcome; nothing beats clean template DNA)
2 µl dNTP mix
1.5 µl each primer
3 µl MgCl2 (50 mM)
0.5 µl Taq (If using Pfu omit MgCl2)
Cover with mineral oil (this is old-style PCR. Most machines today take smaller tubes and have
heated lids so that mineral oil is not necessary).
Primer Design
Make forward and reverse primers with the same melting temperature. Ideally with 50% GC
content. Use a melting temp calculator for this purpose.
For 2 L:
40 ml 0.5 M EDTA
200 ml 10X PBS
QS to 2 L.
Proteinase K Reaction Buffer
0.01 M Tris, pH 7.8
0.05 M EDTA
0.5% SDS
Proteinase K Digestion (Example using Borrelia burgdorferi, for the removal of
surface proteins)
950 µl Borrelia at 1 x 10-9 (1000,000,000) + 50 µl Proteinase K (4 mg/ml stock)
E/E RT 10 min
Stop reaction with 10 µl PMSF (0.1 M)
Potassium Chloride (0.5 M)
9.32 g QS to 250 ml with ddH2O
PCR (example)
73.5 µl H2O
10 µl 10X buffer (supplied with the polymerase)
2 µl DNA (this amount/concentration highly affects outcome; nothing beats clean template DNA)
2 µl dNTP mix
1.5 µl each primer
3 µl MgCl2 (50 mM)
0.5 µl Taq (If using Pfu omit MgCl2)
Cover with mineral oil (this is old-style PCR. Most machines today take smaller tubes and have
heated lids so that mineral oil is not necessary).
Primer Design
Make forward and reverse primers with the same melting temperature. Ideally with 50% GC
content. Use a melting temp calculator for this purpose.
