3 Methods
3.1 Amplification
and Purification
of the Y2H Bait
Constructs
1. Set up two 50 μL PCR reactions to amplify the wild type and
PBM mutant versions of the bait of interest using the forward
and reverse primers described in Subheading 2.1.
2. Start by mixing 1 μL of dNTPs, 2 μL of Forward and Reverse
primers, 50–100 ng of template bait DNA, the volume of PCR
reaction buffer and magnesium chloride recommended by the
DNA polymerase manufacturer, complete with sterile ddH 2 O
water up to 50 μL and finish with the addition of DNA polymerase, as recommended by the manufacturer.
3. Run both PCR reactions according to the DNA polymerase
manufacturer recommendations. A rather low number (typically 18 to 20) of PCR cycles is generally enough when amplifying short DNA fragments.
4. Check the size and abundance of the PCR product by electrophoresis, using a 1–2% agarose gel (depending on the size of
the amplified DNA fragment) and loading 2 μL of the PCR
reaction.
5. Gel-purify 10–20 μL of the PCR products on a new agarose gel
to remove any unreacted primers that may interfere with the
recombination step. To accomplish this step use the reagents
provided with the gel DNA extraction kit and following the
manufacturer instructions. Be aware that small PCR products
(<100 bp) tend not to bind thoroughly to the column, thereby
potentially reducing their recovery after purification.
3.2 Cloning of Bait
Entry Clones in pDONr/
pZeo
®
Here we describe the successive steps for subcloning PCR amplified
bait constructs, as obtained in Subheading 3.1, into the entry clone
vector pDONr/pZEO
®
1. Set up two BP recombination reactions by combining 1–2 μL
of gel-purified PCR fragment, 300 ng of pDONr/pZeo
® , 2 μL
of TE buffer, and 2 μL of BP recombinase
® .
2. Incubate at RT for at least 1 h.
3. Transform the recombination reaction mix in chemically competent bacteria and select transformants on 1Â Zeocinsupplemented LB Agar plates at 37
C.
4. Next day, inoculate a few transformants in 3 mL of 1Â Zeocinsupplemented liquid LB. Incubate overnight at 37
C and
recover the bacterial cells in 1.5 mL microtubes by a 10 min
centrifugation at 16,000 Â g.
5. Purify the DNA clones using a DNA miniprep extraction kit
and following instructions provided by the kit manufacturer.
8
Monica Castro-Cruz et al.
3.1 Amplification
and Purification
of the Y2H Bait
Constructs
1. Set up two 50 μL PCR reactions to amplify the wild type and
PBM mutant versions of the bait of interest using the forward
and reverse primers described in Subheading 2.1.
2. Start by mixing 1 μL of dNTPs, 2 μL of Forward and Reverse
primers, 50–100 ng of template bait DNA, the volume of PCR
reaction buffer and magnesium chloride recommended by the
DNA polymerase manufacturer, complete with sterile ddH 2 O
water up to 50 μL and finish with the addition of DNA polymerase, as recommended by the manufacturer.
3. Run both PCR reactions according to the DNA polymerase
manufacturer recommendations. A rather low number (typically 18 to 20) of PCR cycles is generally enough when amplifying short DNA fragments.
4. Check the size and abundance of the PCR product by electrophoresis, using a 1–2% agarose gel (depending on the size of
the amplified DNA fragment) and loading 2 μL of the PCR
reaction.
5. Gel-purify 10–20 μL of the PCR products on a new agarose gel
to remove any unreacted primers that may interfere with the
recombination step. To accomplish this step use the reagents
provided with the gel DNA extraction kit and following the
manufacturer instructions. Be aware that small PCR products
(<100 bp) tend not to bind thoroughly to the column, thereby
potentially reducing their recovery after purification.
3.2 Cloning of Bait
Entry Clones in pDONr/
pZeo
®
Here we describe the successive steps for subcloning PCR amplified
bait constructs, as obtained in Subheading 3.1, into the entry clone
vector pDONr/pZEO
®
1. Set up two BP recombination reactions by combining 1–2 μL
of gel-purified PCR fragment, 300 ng of pDONr/pZeo
® , 2 μL
of TE buffer, and 2 μL of BP recombinase
® .
2. Incubate at RT for at least 1 h.
3. Transform the recombination reaction mix in chemically competent bacteria and select transformants on 1Â Zeocinsupplemented LB Agar plates at 37
C.
4. Next day, inoculate a few transformants in 3 mL of 1Â Zeocinsupplemented liquid LB. Incubate overnight at 37
C and
recover the bacterial cells in 1.5 mL microtubes by a 10 min
centrifugation at 16,000 Â g.
5. Purify the DNA clones using a DNA miniprep extraction kit
and following instructions provided by the kit manufacturer.
8
Monica Castro-Cruz et al.