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From 2011.igem.org

Notebook: Week 5

Monday

Tuesday Tried absorbance; too messy (see lab notebook, Nanodrop pictures) Fluorescence via fluorimeter indicates GFP is found in supernatant, but not in pellet from lysate Tried running tris tricine gel instead of normal glycine gel

Wednesday Protocols for sequencing (http://medicine.yale.edu/keck/dna/protocols/tube/index.aspx):

- 500-600 ng ds plasmid DNA template

- 2 microliters of 4 micromolar primer

- fill up to 18 microliters water

For today, here are the amounts:

- MBP-His-TEV-AFP Starting Concentration: 135.8 ng/microliter

- malF: 124.7 ng/microliter

- malR: 107.1 ng/microliter

Use 4 microliters of MBP-His-TEV-AFP, which brings us to approximately 550 ng, which is the required amount of plasmid, add 4 microliters to each of two sequencing tubes. Next, I calculated molar concentration of our primers, via http://www.genscript.com/conversion.html:

In 1 microliter of malF, we have 124.7 ng, so to calculate the molarity: 124.7 ng*(10^3 pg/1 ng)*(1 pmol/330 pg)*(1/25 nucleotides) = 15.12 pmol in 1 microliter, or 15.12 micromolar malF; repeat similarly for malR to get 12.98 micromolar malR.

We need 4 micromolar primer, which I made a 10 microliter "stock" solution of, so the calculation is as follows: 15.12 micromolar*(x amount of primer) = 10 microliters*4 micromolar, so x = 2.64 microliters for malF and 3.08 microliters for malR. I added that amount and then filled up to 10 microliters with water. I then took 2 microliters of each stock primer solution and added to the appropriate sequencing tube, then filled up to 18 microliters with water. Finally, I labeled the tubes appropriately (http://medicine.yale.edu/keck/dna/protocols/tube/labeling.aspx).

http://128.36.30.107/fmi/iwp/cgi?-db=keck_dna_seq&-loadframes, go to 'Premix individual tube order'