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.
   1. For the Bs, do the same process using the same 2 ml collection tube/spin column
   2. Do the same for the Ss
      1. Basically, we are combining the DNA for 1-4.
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 ul of Buffer EB (10 mM Tris·Cl, pH 8.5) 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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Day 13: Sequencing DNA

1. Determine how many sequencing reactions you will be sending out, and ask Alain for a purchase order number for Genewiz, a DNA sequencing service.
2. Take as many 8-tube strips as you need, and label them with your initials and a three-digit number, e.g. "PT001, PT002...". Plan and record which sequencing reactions you'll put into which tubes.
3. Into each tube, pipette 8ul of the DNA to be sequenced. If you're premixing your own primer, add 4ul primer (2uM).
   1. Each sequencing reaction should contain only one primer. For each DNA sequence you want to be analyzed, you should have two sequencing reactions, one with forward primer and one with reverse primer.
   2. You may leave out primer if you are using a vector with annealing sites for universal primers. For example, the Topo vector has M13F and M13R primer annealing sites flanking the cloning site, and Genewiz can add those primers for you.
4. Snap the tubes closed, wrap the strip in parafilm, and seal in a Ziplock bag.
5. Log into www.genewiz.com with Alain's email aviel@fas, and password ******.
6. Click "Place DNA Sequencing Order".
7. Fill in "Number of Reactions", select "Premix", and select yes or no for "Same Day Service". #Click "Submit".
8. Fill in the "PO Number". Fill in the sizes of your DNA samples to be sequenced. Select "DNA Type", usually plasmid or PCR. If you've already added primer, under Primer, select "Already added", and if you want Genewiz to add universal primers, select the proper primer "to be added". #Review the order, and click "Submit."
   1. If you are sequencing an insert within a plasmid, fill in the size of the entire plasmid plus insert.
9. Print out two copies, one for Genewiz and one for yourself. Fold up the copy for Genewiz, and place into the Ziplock bag with the tubes. Drop off the bag in the dropbox at Conant 113 before 4pm Monday-Friday.
   1. The entrance to Conant is in the walkway between Lowell Lecture Hall and Fairchild.
      1. A Big Thanks to Perry for this Sequencing Protocol

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Protocol Recorded by Kevin Shee