Aim: to get DNA expression in E. coli.

• Remove chemically competent E. coli cells from the -80oC freezer and thaw on ice.
• Take 1-2 µL of DNA and transfer into a clean 1.5 mL tube.
• Add 50 µL of chemically competent E. coli to the DNA and incubate on ice for 30 mins.
• Preheat water bath to 42oC.
• Heat shock the DNA and E. coli tube for 30-60 sec (not more than 60 sec).
• Transfer back onto ice for 5 mins.
• Add 250-500 µL of LB broth to the tube and incubate at 370C with shaking for 2 hrs.
• Spread plate 100 µL onto plates containing the relevant antibiotics and incubate over night at 37oC.

Aim: to determine the size (bp) of the DNA expressed by the colony picked. This helps us to check that the construct is what we expected.

• Create an overnight culture- Pick a single colony from a freshly streaked selective plate and inoculate a culture of 1–5 ml LB medium containing the appropriate selective antibiotic. Incubate for 12–16 h at 37°C with shaking.
• Centrifuge 1-5ml of overnight culture at >8000 for 3 minutes in an Eppendorf tube to form a bacterial pellet; discard the supernatant.
• Re-suspend bacterial pellet in 250µl of P1 Buffer (kept at <5oC).
• Add 250µl of P2 Buffer and invert 4-6 times to mix thoroughly. This reaction is left for no longer than 5 minutes before completing the next step.
• Add 350µl of N3 Buffer and invert 4-6 times to mix thoroughly.
• Centrifuge for 10 minutes at 13,000rpm
• Decant supernatant into a spin column, centrifuge for 30-60 seconds, and discard the flow-through.
• Add 750µl of PE Buffer to the spin column and centrifuge for 30-60 seconds to wash the DNA. Discard the flow-through and centrifuge for a further 1 minute to remove any remaining buffer.
• Remove the top section of the spin column and place in a clean 1.5ml Eppendorf tube. Add 50µl of EB Buffer to the column; let it stand for 1 minute before centrifuging for a further 1 minute to elute the DNA.

• Add 5µl of DNA sample at a concentration of 80-100ng/µl of Plasmid DNA (Diluting with sterile water if Plasmid DNA is at a concentration greater than 100ng/µl) to two separate 1.5 ml Eppendorf tubes.
• Add 5µl of primer 1 (Sense) to the first tube at a total concentration of 5µM (5pmol/µl) and 5µl of primer 2 (AntiSense) to the second tube (at a total concentration of 5µM (5pmol/µl)
• Send off both 10µl samples for sequencing.