3 Materials and Methods
3.3 PCR Applications
3.3.1 PCR-Cycler
A tool commonly applied during this thesis was the polymerase chain reaction (PCR). Several PCR Cyclers were used, based on machine availability:
• Eppendorf Mastercycler gradient and Eppendorf Mastercycler personal, (Eppendorf GmbH, Hamburg, D),
• PTC-100 ™ (MJ Research, Inc., Watertown, Mass., USA) and
• MiniCycler™ (MJ Research, Inc.), without top heating.
3.3.2 Chemicals for PCR Applications
For PCR applications, Millipore® water was used as diluting agent. The dNTPs (MBI Fermentas, St.Leon Rot, D) were mixed to obtain a 10-fold concentration. The buffer was mixed to obtain 5-fold concentration already containing MgCl2. To prevent the mixture from evaporating, an oil layer was applied when using the MiniCycler ™ without top heating or when the reaction mixture was 20 µl or less (except for sequencing reactions). In this case, 20 µl mineral oil (Sigma Aldrich, Steinheim, D) was deposited atop the reaction mix.
A standard PCR reaction contained:
1x Reaction buffer (50 mM KCl, 1.5 mM MgCl2, 10 mM Tris, pH 9.0) 0.2 mM dNTPs (Equal ratio blend of dATP, dGTP, dCTP, dTTP) 0.5 µM upper primer
0.5 µM lower primer 0.02 U/µl Taq polymerase 20 ng Template DNA
Millipore H2O up to the final volume
The elongation time was chosen depending on the length of the template DNA. For template DNA up to 1 kb, the program Universal 1.5 was chosen, and for DNA up to 2 kB (or if the length was unclear as for plaque PCR), Universal 2.0 was applied.
For sequencing reactions either an upper or a lower primer (0.2 mM) was used. The reaction was performed using 3 µl of the BigDye Terminator Cycle Sequencing Ready Reaction Mix 1.0 or 2.0 Kit (Perkin Elmer, Applied Biosystems Darmstadt, D) including: A-Dye Terminator labeled with dichloro[R6G]; C-Dye Terminator labeled with dichloro[ROX]; G-Dye Terminator labeled with dichloro[R110]; T-G-Dye Terminator labeled with dichloro[TAMRA]; Deoxynucleoside triphosphates (dATP, dCTP, dITP, dUTP); AmpliTaq®
DNA Polymerase, FS, with thermally stable pyrophosphatase; magnesium chloride, and as Reaction buffer, Tris-HCl buffer, pH 9.0. The final volume was 10 µl, containing 20 ng template DNA.
3.3.3 PCR Programs
3 different PCR programs were used during this thesis. The parameters used for each program are listed below:
Table 3-14 PCR Program Parameters PCR Program
Process steps Universal 1.5 Universal 2.0 GATC Seq.
1. First denaturing step 2 min at 94°C 2 min at 94°C 1.5 min at 95°C
2. Elongation cycle 29 cycles 29 cycles 25 cycles
a. Denaturing 30 sec at 94°C 30 sec at 94°C 1.5 min at 95°C b. Annealing 30 sec at 60°C 30 sec at 60°C 30 sec at 50°C c. Elongation 1.5 min at 72°C 2 min at 72°C 4 min at 60°C 3. Last elongation 5 min at 72°C 5 min at 72°C
-4. Cooling phase ∞ 4°C ∞ 4°C ∞ 2°C
3.3.4 Primers
The primers used during this thesis are described in the following two tables:
Table 3-15 List of Primers for General Use
Name Sequence (5´→ 3´) Used for
GT10F GAG CAA GTT CAG CCT GG λGT10
GT10R CTT ATG AGT ATT TCT TCC AGG GTA λGT10
pET T7n ACC CCT CAA GAC CCG TTT AG pET28a
pET term n ACC CCT CAA GAC CCG TTT AG pET28a
pDR195up TCT CTT TCT TTC CTA TAA CAC CA pDR195
pDR195rev AAC CTT GAT TGG AGA CTT GAC pDR195
PIG5-1 GTT GGT CTC CAT CAA AGC PIG5
PIG5-2 CGA ATT TTT GAG CAC CAT CC PIG5
PIG7-1 CGC AAC TCT ACA ACT CAA C PIG7
PIG7-2 AAG TGC GAC CAA GGT AAG PIG7
PIG7-3 TGC GGA AGT GTG ATT AGA AGG PIG7
PIG7-10 5’ CAA ACC TTC GCT TAC TTT TCA C PIG7
PIG7-11 3’ CGG GTA TGA TGA AAT CCC PIG7
PIG9-2 CTG TGG CTT TCT TTG GTC PIG9
PIG9-3 AGC TTG AAC AAC GCA GTC C PIG9
PIG9-4 TGA GGA CTC TTG GCT ATG PIG9
PIG23-1 GTG CTT GTT GTA TTC TTC C PIG23
These primers were used for applications such as plaque PCR, amplification of cloned inserts and sequencing reactions. They were also applied to generate DIG-labeled probes for Northern and Southern blotting.
In order to affix a restriction site to cDNA fragments for cloning into vectors, special primers were designed. In addition to the inverse target sequence, recognition sites for chosen restriction enzymes with a number of protective bases flanking the 5’-end were manufactured.
These extra nucleotides were required because some restriction enzymes cleave DNA poorly when their recognition sites are located at the ends of DNA fragments.
The above-mentioned special primers are described in the following table:
Table 3-16 List of Primers for Restriction Site Affixation to cDNA Fragments
Name Sequence (5´→ 3´) Restriction
site
Used for
PIG5-Cleavage
GCT AGT ACA TAT GGC CAT CCC TAT GGT GGC CAA TC NdeI PIG5
PIG5-5’ GTC GCG GAT CCG TTA TTA GGC AAA TTG TGA C BamHI PIG5
PIG5-3’ GTC GAC GGA GCT CGC CCG TGG ACA ATC ATG SacI PIG5
PIG7-Cleavage
CGA CCT CCA TAT GCA ACT AGT AGG CTC AGA TGT G NdeI PIG7
PIG9-Cleavage
GCT AGT CCA TAT GCC TCT GAT TAC AGC CGA AAC NdeI PIG9
PIG9Mitte CAG ACC ACA TAT GGC CAC AGA GAA AGG CAC TAA G NdeI PIG9 PIG9-3’short GCT GAC GGA GCT CCC ATC GTG CTT GCC TTG AG SacI PIG9 PIG9-3’long GTC GAC GGA GCT CCA AAT CAA GGC TTA GAC SacI PIG9
PIG9-5’ GTC GCG GAT CCA TTA CAG CCG AAA CAG CAC BamHI PIG9
PIG14-5’ GTC GCG GAT CCG TAT CTC GCC TTG CTC CTC G BamHI PIG14 PIG14-3’ GTC GAC GGA GCT CGT CAT TTG GTG GAC TCA TAG SacI PIG14 PIG23-5’ GAA TTC GAG CTC ATG GAT CAC CCG AAA CAA C SacI PIG23
PIG23-3’ GGT GCT CGA GCA GGT TTG TTA GGC GTG GTC XhoI PIG23
PIG14-Cleavage
GAT CCT GCA TAT GAA CGC AAC TGG CCC AAG C NdeI PIG14
PIG15-Cleavage
GCC AGT CCA TAT GGT TAG ACC TTA CAA TCG TAT CCC NdeI PIG15
PIG23-Cleavage
GCC AGT CCA TAT GTT TTC TCA GGC TCC AGT GTT A NdeI PIG23
For reasons of convenience and economy, the abovementioned special primers were also applied for control PCRs, as well as to generate DIG-labeled probes for Northern and Southern blotting.
3.3.5 PCR Product Purification
PCR products were purified using the Qiaquick PCR Purification Kit (Qiagen GmbH, Hilden, D) or the E.Z.N.A® Cycle-Pure Kit (Peqlab, Biotechnologie GmbH, Erlangen, D).
Both spin column purifications kits were used as specified in the manufacturers manuals.
When using the Qiagen kit, the suggested optional washing step with 35% guanidine hydrochloride proved to be essential in removing all primers.
3.3.6 DIG Non-Radioactive Nucleic Acid Labeling
Digoxigenin-11-dUTP (DIG-dUTP) was incorporated into the DNA probe by Taq DNA polymerase during PCR in order to identify DNA or RNA on a Southern- respective Northern blot. Preferentially, the primers were chosen to replicate only cDNA sections and to exclude the λGT10 phage. In some cases (PIG7, PIG14 and PIG15) it was unavoidable to include short sections of the phage in the DIG-labeled DNA probe in order to obtain a sufficiently large probe.
Table 3-17 10x DIG-dNTP Mix for DIG-Labeled Probes Nucleotide Final concentration Manufacturer
dGTP 2.00 mM Boehringer Mannheim, Mannheim, D
dCTP 2.00 mM Boehringer Mannheim, Mannheim, D
dATP 2.00 mM Boehringer Mannheim, Mannheim, D
dTTP 1.33 mM Boehringer Mannheim, Mannheim, D
DIG-dUTP 0.67 mM Boehringer Mannheim, Mannheim, D
Table 3-18 Primer Combinations for DIG-Labeled Probes DIG-probe for 5’-primer 3’-primer Template
PIG5 PIG5-5’ PIG5-3’ H322
PIG7 PIG7-10 PIG7-11 PDR195:PIG7 (originating from H9)
PIG9 PIG9-5’ PIG9-3’ H62
PIG14 GT10R GT10F H1408
PIG15 GT10R PIG15-2 H1309
PIG23 PIG23-5’ PIG23-3’ H1063
The template DNA for the DIG-labeled probes originated from the haustorial EST project.