IGEM:Harvard/2007/week 2

Day 13: Nucleotide Removal (6/25/07)

1. Add 10 volumes of Buffer PN to 1 volume of random library extensions (single primer) and mix.
   1. B1, B2, B3, B4 and S1, S2, S3, S4 (about 50 ul each)
2. Place a QIAquick spin column in a provided 2 ml collection tube.
3. To bind DNA, apply the sample to the QIAquick column and centrifuge for 1 min at 6000 rpm.
4. Discard the flow-through and place QIAquick column back into the same tube.
5. To wash QIAquick column, add 750 µl of Buffer PE and centrifuge for 1 min at 6000 rpm.
6. Discard the flow-through and place the QIAquick column back in the same tube, which should be empty. Centrifuge for an additional 1 min at 13,000 rpm (~17,900 x g).
7. Place the QIAquick column in a clean 1.5 ml microcentrifuge tube.
8. To elute DNA, add 100–200 µl of Buffer EB (10 mM Tris·Cl, pH 8.5) or H2O to the center of the QIAquick membrane and centrifuge the column for 1 min at 13,000 rpm (~17,900 x g). Alternatively, for increased DNA concentration, add 30–50 µl elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge.

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Day 13: Transformation protocol of ligated DNA (6/25/07)

1. Thaw the 4 tubes of cells on ice and mix gently to ensure that the cells are evenly suspended. We will be using the following DNA:
   1. OmpA1/G1/S2
   2. OmpA1/G1/S
   3. OmpA2/G2/B
   4. OmpA2/G2/S
2. Add 5 µl of the DNA solution directly to the cells. Stir gently to mix.
3. Place the tubes on ice for a little more than 5 minutes
4. Heat the tubes for about 45 seconds in a 42°C water bath; do not shake
5. Place on ice for 2 minutes
6. Add 250 µl of room temperature SOC Medium to each tube
7. Shake at 37°C (300 rpm) for more than 30 minutes.
8. Light the bunsen burner
9. Using two sets of petri dishes containing LB agar w/ Kanamycin (1 w/ 5 µl of cells and one w/ 50 µl of cells):
   1. 5 µl: pipette 5 µl of cells with 45 µl of SOC onto the agar mix
   2. 50 µl: pipette 50 µl of cells onto the agar mix.
10. For each plate, use sterile beads to spread the bacteria over the mix.
11. Afterwards, turn the plates upside down and incubate at 37°C

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