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Team:Stanford/Notebook/Lab Work/Week 7
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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 |
Karina's Notebook
To 3A or not to 3A... that is the question.
Amount of DNA in ligation is key!
- For 10 uL ligation, never put less than 150 ng of backbone
Running low on stock of RSIDs and sRNAs, need to make more in order to proceed with assembly.
Also need to digest pBad and pLux
Make Antibiotic Plates
Recipe (per 500 mL bottle)
5 g Bacto-Peptone
2.5 g Yeast Extract
2.5 g NaCl
6 g Agar
- measure out and add to each bottle. Add 450 mL millipore H20 and shake vigorously.
- Total Volume --> 500 mL
- Adhere autoclave tabe, LOOSEN CAP, and set in autoclave tray
- Start program (autoclave liquid)
- Once program ends, use GLOVES to remove bottles and let cool. Once cool enough to keep your hand on the bottle for at least 10 s, add antibiotic.
Antibiotic Concentrations:
A 50 ug/mL
K 20 ug/mL
C 34 ug/mL
T 20 ug/mL
From a 1000x stock, add .5 mL antibiotic to 500 mL solution. Pour plates and let solidify over night.
Sequencing Analysis
GFP looks good! RFP has wierd, additional bases at the very end of RFP but before suffix. It could be part of the BioBrick part, will send E1010 for sequencing tomorrow just to double check.
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
Karina's Notebook
Goal: 3A Assembly
We don't have enough DNA, need to redo PCR assembly. Upscaling:
10 uL of each primer
30 uL supermix
3 reactions of each product. 10 cycle PCR.
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"
- different starting DNA concentrations: 0.25X, 0.5X, 1X, 2X
- different annealing temperatures: 50oC, 55oC, 60oC
- 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
- thermal cylcler program: FLK
- run on 2% gel at 90V for 30 minutes
Step 2: try different annealing temperatures
- use 1 uL each primer; 8 uL PCR SuperMix in each reaction (3 + 3 + 24 in one tube, then 10 uL aliquoted to other 2 tubes)
- cycling (FLK program) modified to include a gradient at the annealing step: 50oC to 62oC
- row H = 50oC, row E = 54.8oC, row C = 59.6oC
