Team:Stanford/Notebook/Lab Work/Week 4

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7/19 Monday

Laura's Notebook

set up the following ligations from the gel extractions done by me, Karina, and Francisco on Friday, 7/14/10)

• Francisco ran and imaged diagnostic gel on Friday

• normally run for 10 minutes at room temperature, overnight this time (started at 11:30 am)

Nanodrop data (deemed unreliable based on 260/230, possibly due do residual EtBr contamination)

Karina's Notebook

Goal: Laura will ligate GFP and RFP and ligate them to terminators. We received our RSID + RBS oligos in the mail, so I will work on the PCR. I first need to make freezer stock and working stock of oligos. Won't start PCR until after lunch because we'll leave them overnight with Chris' PCR reactions.

Make Tris-HCl
Need .01 L of 10mM Tris-HCl solution. So, add 15.76 mg Tris-HCl to 10 mL H20.

• Hard to weigh out 15.8 mg, so instead got to 18.3 mg.
• Determined that need to add this to 11.6 mL H20
  

Make Freezer Stock of Oligos
Want 100 uM solution of Tris-HCl solution

• Amount of Tris-HCl to add depends on how much of the oligo's we received.

Make Working Stock
Want a 10uM working stock solution

• 900 uL water + 100 uL Freezer Stock Solution
  
  

PCR Assembly
PCR Protocol calls for:
1.25 uL reverse primer
1.25 uL forward primer
DNA Template
50 uL PCR supermix

• For PCR Assembly, do not need DNA template. Also, add 4 times as much of each primer for PCR assembly.
  

Revised Recipe
5 uL Forward Primer
5 uL Reverse Primer
40 uL PCR Supermix

• we'll be using awesome PCR supermix- KEEP ON ICE
• Ran PCR with Chris, left running overnight

7/20 Tuesday

Laura's Notebook

helped Alex with preparation of competent cells

• see protocol: [http://openwetware.org/wiki/Stanford/BIOE44:Module_1:Day3 Preparing Electrocompetent Cells]

ran diagnostic gel for PCR-assembled DNA (Karina set up PCR rxns yesterday)

• order on gel:

1. 100 bp ladder
2. RSID1/RBS
3. RSID2/RBS

Karina's Notebook

Goals: Run gel of PCR Assembly Product, gel extraction, digest, then run PCR clean up.

Make Gel

• Chris made me an 0.8% agarose gel
  
  

Load Gel

• added 10 uL loading dye directly into PCR tubes
• loaded 40 uL of each sample into wells
  
  

Gel Results
[insert picture here]

Analysis/Observations
Gel looked a little streaky to me, but we really only need the brightest bands (as circled). For next time:

• use 100 bp ladder (instead of 1 kb)
• Don't even bother with gel extraction after running a PCR. It is highly inefficient and a lot of DNA will be lost. Instead, run a a diagnostic gel to check that PCR assembly worked but then proceed to run a PCR cleanup and then digest.
  
  

Extraction
Extracted 1A, 1B, 2B, and 2C from the gel.

• extracted and incubated each of the samples separately. To combine, loaded 1A and 1B into one spin column and 2B and 2C into another. Loaded up to 800 uL, spun down, loaded, then spun again. Then continued to follow [http://molecool.wustl.edu/krolllab/Kroll_Lab_Protocols/Molecular%20Biology%20protocols/Cloning%20protocols%20folder/Gel%20extraction-Qiagen.pdf procedure].
  
  

PCR Assembly
Set up another PCR assembly of RSID 1 and RSID 2 so that we have samples that have not been gel extracted. Ran another three samples of each. Will let run overnight.

• Primers were designed to anneal at 55º, but set up a temperature gradient to test annealing at other temperatures.

7/21 Wednesday

Laura's Notebook

today's digestions:

• RSID1, RSID2 (gel extracted from first PCR done by Karina)- digest with XbaI, PstI
  • run overnight; started at 11:30am
• promoters within backbones (I0500-from Greg's box, and F2620-from Chris' box)- digest with SpeI, then PstI
  • SpeI digest run for 3 hours, then heat killed 20 min. at 80oC, then PstI added overnight (to reduce enzyme competition for sites on the DNA, since recognition sequences are very close to each other)

Recipe:

Kairna's Notebook

Goal: Left Laura to gel extract previous PCR product as I worked on the new PCR product. Today, must make and run a diagnostic gel, PCR clean up, then digest RSID 1 and RSID 2.

Make TAE
Need a 1 L solution; we have 50x TAE stock

• 20 mL 50x TAE + 980 mL H20
  
  

Make Gel
Want a 2% agarose gel because oligos are short (~220 bp).

• 1 g agarose + 50 mL TAE
• 10 uL EtBr
  

Also, made Chris an 0.8% agarose gel (to repay him for yesterday).

Load Gel
10 uL 100 bp ladder
1 uL dye + 1 uL DNA

Results
[link here]

Great! No difference in the annealing temperatures. All bands are clear and bright, can use all three samples for restriction digests.

PCR Cleanup
Used Qiagen MinElute PCR Purification Kit Protocol.

• 50 uL reaction, therefore use 250 uL buffer PB
• Followed protocol and eluted in 10 uL water.
  
  

Restriction Digest
Cut at X and P

Recipe

• 10 uL DNA
• 5 uL NEBuffer 2
• 5 uL BSA
• 1 uL Xba
• 1 uL Pst
• 28 uL H20

Total volume: 50 uL

Let run overnight in 37º waterbath