Team:Stanford/Notebook/Lab Work/Week 7

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Contents

8/9 Monday

Laura's Notebook

  • helped Alex NanoDrop some of his samples (concentrations also written on tops of tubes)
260/280 260/230 ng/uL
sGFP mDNA 1.71 1.01 320.3
sGFP mDNA s 1.91 1.41 220.7
F2620 1.10 0.87 45.0
F2620 s 1.52 0.88 53.1
I5 1.90 1.06 165.8
I5s 1.98 1.22 113.9
I4 1.73 0.95 384.2
I0500 1.86 0.89 67.7
C1 1.77 0.80 83.0
C2 1.83 0.83 86.6
C3 1.95 1.09 75.7

8/10 Tuesday

Laura's Notebook

set up colony PCR for single colony from pLUX-sRNA1C ligation/transformation:

  • 9 uL supermix, 0.5 uL of each primer (VF, VR)
  • cycling program as before: COLPCR72


3pm meeting with Drew, Rayka about device names, parameters

8/11 Wednesday

Laura's Notebook

miniprepped pBAD, pLUX

  • used Promega kit
  • combined 2 liquid cultures for each into 1 tube (when pelleting cells)
  • eluted in 50 uL H2O (from kit)


set up colony PCR (same as yesterday; tube dried up in machine)


troubleshooting PCR stitching reactions (prior to ligations)- recommendations from Christina:

  • each "step:" try RSID2, sRNA1C; use best result to continue to next "step"
  1. different starting DNA concentrations: 0.25X, 0.5X, 1X, 2X
  2. different annealing temperatures: 50oC, 55oC, 60oC
  3. cycle numbers: 5, 10, 15, 20


Step 1: try different DNA concentrations

  • Francisco set up tubes and added PCR SuperMix (10 uL reactions)
  • I added primers to 1 series of 4, he did the other
  • run on 2% gel at 90V for 30 minutes

8/12 Thursday

8/13 Friday