Protocols · Benchling

Materials

Make master plates by dividing each LB agar plate in 6 numbered sections, continue numbering on the next plate.

Thaw solutions for PCR master mix on ice.

Suspend primers to concentration 100µM (see specification sheet)

Gently vortex and briefly centrifuge all solutions after thawing.

Make dNTP Mix by adding equal volumes of each dNTP (100mM) to a sterile tube.

Make a master mix: enough for wanted number of 20µL reactions + 1 (for margins)

a) Number of 20µL reactions: 1 per clone + 1 negative (no template DNA)

b) Recipe for 20µL reaction, mix thoroughly:

Solution	µL
10x Taq Buffer with KCl	2
Each dNTP (100mM each)	0.04 OF EACH (0.2 mM final conc of each)
Forward primer (100 µM)	0.06 (0.3 µM final conc)
Reverse primer (100 µM)	0.06 (0.3 µM final conc)
MgCl2 (25 mM)	1.2 (1.5 mM final conc)
Taq DNA Polymerase (5 U/µL)	0.1 (0.5 U)
Nuclease free water	16.42 (to reach 20 µL)

Place thin-walled PCR tubes on ice and add 20µL master mix to each

Pick a single colony (clone) from a transformant plate with a sterile inoculation loop and streak on the center of an empty (but labelled) section of a master plate. Rinse the same loop in a PCR tube with master mix (20µL reaction).

a) Repeat for each clone, ~3 clones per successful transformant plate. Remember to take from the plate with intact plasmid without insert.

b) Incubate master plates at 37°C until step 12.

Gently vortex the PCR tubes and spin down

Perform PCR:

Step	Duration	Temperature	Number of cycles
Pre-heating	10 min	95 °C	1
Denature	30 sec	95 °C	30
Annealing	30 sec	61 - 5 °C (Depends on primers)=56°C	30
Elongation	1 min/kbp	72 °C	30
Final elongation	10 min	72 °C	1

Analyse on gel to detect presence of PCR product of expected length.

For each clone which yielded the correct PCR product: inoculate overnight culture using the colony from the correct section of the master plate after it has had time to incubate.

Store master plates in the fridge or cold room.

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