User:Howard Boland/Notebook/Art from Synthetic Biology/2010/09/16

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Nanodrop

Based on the confirmed digest -link needed- I did a nanodrop of the 3 samples in preparation for sequencing.

  1. Sample: pUA66katE-10092010-Green: 27.5ng/ul
  2. Sample: pUA66katE-14092010-Blue: 17.8ng/ul
  3. Sample: pUA66katE-14092010-Red: 26.8ng/ul

Sequencing preparation

In order to prepare my samples for sequencing the following concentrations were prepared

Requirements:

  1. Primers 2-5mol, 6ul
  2. Plasmid 100ng/ul, 10ul

Dilution calculation: 48nmol in 480nl of RNAse free water gives 0.1nmol/ul = 100 pmol/ul thus to obtain the concentration for the sequencing we will need 6ul x 4pmol/ul) / 100pmol/ul = 0.24ul of primer in 5.76ul of water (total 6ul)

I prepared both my primers as follows: μL

  1. 0.96ul Primer (4x0.24ul)
  2. 23.04ul (4x5.76ul)

I prepared my vector by taking 20ul:

  1. 20ul of Vector pUA66katE-14092010-Red, 26.8ng/ul

The order sheets and specifications where attached with the samples and sent to the Wolfson institute for sequencing.

End Overnight Growth

I took at the two samples from the overnight growth

  1. Glycerol stock
  2. Centrifuge for 10 minutes
  3. One bottle went for miniprep the other was stored at -20°C


Miniprep

I set up a miniprep using one of today's overnight growth and one from the four prepared yesterday. The samples extracted using QIAgen Miniprepp kit and eluted in 30μL water.

Digestion XhoI/BamHI

The extracted plasmids (two samples) from the miniprep were each immediatly digested using

  1. 0.5ul of BSA
  2. 5ul of NEB#3
  3. 1ul XhoI
  4. 1ul BamHI
  5. 12.5ul Water

Agarose Gel

The digestions where run on a 2% Agarose Gel

  1. Lane 1: 10ul, 100bp NEB Ladder
  2. Lane 2: 50ul Digestion, pUA66katE XhoI/BamHI
  3. Lane 3: 50ul Digestion, pUA66katE XhoI/BamHI