Team:Stanford/Notebook/Lab Work/Week 4
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'''PCR Assembly '''<br/> | '''PCR Assembly '''<br/> | ||
- | PCR | + | PCR Protocol calls for:<br/> |
1.25 uL reverse primer <br/> | 1.25 uL reverse primer <br/> | ||
1.25 uL forward primer <br/> | 1.25 uL forward primer <br/> | ||
+ | DNA Template <br/> | ||
50 uL PCR supermix<br/> | 50 uL PCR supermix<br/> | ||
- | * | + | *For PCR Assembly, do not need DNA template. Also, add 4 times as much of each primer for PCR assembly. <br/> |
''Revised Recipe''<br/> | ''Revised Recipe''<br/> | ||
5 uL Forward Primer <br/> | 5 uL Forward Primer <br/> |
Revision as of 17:40, 22 July 2010
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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
Ligation Recipe | |
---|---|
dH2O | none |
vector (with terminators attached) | 5.0 uL |
insert (RFP or GFP) | 12.0 uL |
10X buffer | 2.0 uL |
T4 ligase | 1.0 uL |
- 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)
part | 260/280 | 260/230 | ng/uL |
vector/terminator | 1.77 | 0.03 | 19.1 |
RFP | 1.86 | 0.02 | 16.2 |
GFP | 1.83 | 0.02 | 14.8 |
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.
amount (nmol) | mass (mg) | amount of Tris to add (uL) | |
RSID 1 + RBS Forward | 70.2 | 1.82 | 702 |
RSID 1 + RBS Reverse | 76.7 | 1.85 | 767 |
RSID 2 + RBS Forward | 81.1 | 2.22 | 811 |
RSID 2 + RBS Reverse | 83.5 | 2.09 | 835 |
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:
- 100 bp ladder
- RSID1/RBS
- 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.
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:
component | amount (uL) |
DNA | 12.0 |
H2O | 26.0 |
10X NEB buffer #2 | 5.0 |
10 X BSA | 5.0 |
each enzyme | 1.0 (2.0 total) |