Trouble shooting for PCR

Hypotheses in order of frequency:
A. Pilot error hypothesis
B. Template dilution hypothesis
C. Temperature errors hypothesis
D. Unique template hypothesis
E. Buffer problems hypothesis
F. Bad dNTPs hypothesis
G. Bad primers hypothesis
H. Bad enzyme hypothesis
I. Bad karma hypothesis

Standard PCR protocol

Materials:

sterile water

10X amplification buffer with 15mM MgCl2

10 mM dNTP

50 μM oligonucleotide primer 1

50 μM oligonucleotide primer 2

5 unit/μl Taq Polymerase

template DNA (1 μg genomic DNA, 0.1-1 ng plasmid DNA) in 10 μl

mineral oil (for thermocyclers without a heated lid
1. Combine the following for each reaction (on ice) in a 0.2 or 0.5 ml tube:
10X PCR buffer 10 μl
Primer 1 1 μl
Primer 2 1 μl
dNTP 2 μl
template DNA and water 85.5 μl
Taq Polymerase 0.5 μl
2. Prepare a control reaction with no template DNA and an additional 10 μl of sterile water.
3. If the thermocycler does not have a heated lid, add 70-100 μl mineral oil (or 2 drops of silicone oil) to each reaction.
4. Place tubes in a thermal cycler preheated to 94 degrees C.
5. Run the following program:
94 degrees C 1 min
55 degrees C 1 min or annealing temperature appropriate for particular primer pair72 degrees C 1 min (if product is <500>500 bp)
for 30 cycles.
Program a final extension at 72 degrees C for 7 min.

DNA protocol (2)

Isolation of DNA from buccal swabs
Materials
50 mM NaOH
1M Tris.Cl, pH 6.5
70% ethanol
CYTo-Pak with Cyto-soft brush (CP-5B; Medical packaging) or Caliber-Cotton-Tipped Apllicator swab (size 6 in; Allegiance Healthcare)
95 degree heating block

Cytogenetics protocol

Culture and metaphase harvest of peripheral blood
Materials
Heparinized whole blood obtained via Vacutainer (Becton Dickinson) or syringe with preservative-free sodium heparin (25 U/ml)

Complete RPMI/10% FBS medium containing 50 microgram/ml gentamycin sulfate in place of penicillin and streptomycin.

100Xphytohemagglutinin-M (PHA; Life Technologies), reconstituted in sterile deionized water (store at 4 degree celcius)

10 microM methotrexate (optional)

1 mM thymidine (optional)

10 microgram/ml Colcemid (Life Technologies)

75 mM KCl (store < or equal 2 weeks at room temperature)

Fixative 3:1 (v/v) HPLC-grade absolute methanol/glacial acetic acid (prepare fresh)

15 ml sterile disposable conical polypropylene centrifuge tubes (do not use polystyrene)

TB syringe with 21-G needle (VWR Scientific; do not use preattached 25-G needle)

RNA isolation protocol (1)

RNA isolation by the rapid Guanidinium method
Materials
Mononuclear cell pellet isolated from 1 to 2 ml bone marrow or 5 ml whole blood
HBSS without Ca or Mg
RNA isolation solution I (25 g guanidinium thiocyanate (4 M final), 29.3 ml H2O, 1.76 ml 0.75 M sodium citrate, pH7.0 (25 mM final) , 2.64 ml 10% (w/v) N-lauroylsarcosine (0.5% final) dissolve at 65 degree celcius, store at room temperature (stable > or equal 3 months)
Do not inhale quanidinium thiocyanate and wear gloves when handling it, Do not expose guanidinium thiocyanate to acid conditions, because this may produce cyanide gas.
2 M sodium acetate, pH 4
Buffered phenol
24:1 (v/v) chloroform/isoamyl alcohol
100% and 70% (v/v) ethanol
RNA isolation solution II (10 ml RNA isolation solution I, 72 microlitre 2-mercaptoethanol (0.1 M final) Make fresh before each extraction)
DEPC-treated H2O

DNA isolation protocol (1)

Isolation of Genomic DNA
DNA isolation from whole blood
Materials
10 ml fresh or thawed frozen whole blood
NKM buffer, 4 degree celcius
Resuspension buffer
10 X TEN solution
2 mg/ml proteinase K
10% (w/v) SDS
Buffered phenol
25:24:1 (v/v) phenol/chloroform/isoamyl alcohol
24:1 (v/v) chloroform/isoamyl alcohol
TE buffer, pH 8.0
Dailysis buffer
Refrigerated centrifuge
Orbital shaker
Dialysis tubing (MWCO 50000)
1.5 ml storage vials