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Team:Hawaii/Notebook/2008-07-28
From 2008.igem.org
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| - | :* PCR reaction to retrieve ccdB gene and BioBrick MCS from | + | :* PCR reaction to retrieve ccdB gene and BioBrick MCS from pSB1A3. |
:* Ran on gel to verify PCR product. | :* Ran on gel to verify PCR product. | ||
:* Gel purified PCR product. | :* Gel purified PCR product. | ||
Revision as of 06:08, 29 July 2008
| Projects | Events | Resources | ||
|---|---|---|---|---|
| Sponsors | Experiments | Milestones | Protocols | |
| Notebook (t) | Meetings (t) |
Things we did today
Wetlab work
Transformation
- Margaret
- Transformation of I14032 into Db3.1
Colony PCR
- Margaret
- Verifying the inserts of two I51020 (the base vector) and one colony of pSB1A7 (an insulated vector with high copy #)
PCR amplification of pRL1383a
- Margaret
- The omega region from pSMC121 and the rep/mob region are giving primer dimers and misamplification respectively. I am trying a gradient PCR from 49°C to 55°C to see if this will fix the problem.
PCR for BB-pRL1383a construction/verification
- Grace
- PCR of BB-pRL1383a (from plasmid prep) using VF2 & VR_B0034 primers to verify B0034 insert and MCS replacement
- PCR of B0034 with HindIII and BamHI primers in case we need to redo BB-pRL1383a construction
- Maintained everything on ice this time
- Used 0.5μl plasmid in PCR reaction
- Annealing at 55C
- Extension for 30 sec. at 72C
- Gel returned 3 bands + huge smear for B0034. Smear = lots of correct product. What are the three other bands (~120bp, ~170bp, ~200bp)?
- Gel gave two bands for BB-pRL1383a (~170bp, ~300bp)
- Will redo BB-pRL1383a construction using ccdB gene as insert/MCS
Plasmid preps
- Grace
- Resuspended plasmid preps in 30 μl TE and checked concentrations on nanodrop spectrometer
- nir+B0034 = 13.6 ng/μl
- GFP+B0024 = 9 ng/μl
- GFPf+B0024= 7.1 ng/μl
- BB-pRL1383a = 166.3 ng/μl
- Restriction digested nir+B0034 plasmid prep to verify insert (GFP and GFPf constructs were verified last week)
- Digested w/ EcoRI and SpeI so we can extract from gel if insert is correct
- Incubated at 37C for 2 hours
- Band way too big 1000+bp
- Need to redo
GFP(f) + tt constructs
- Grace
- Restriction digested GFP and GFPf w/ EcoRI and SpeI in 50 μl reactions for 3 hours
- Restriction digested B0024 (tt) with XbaI then EcoRI for 3 hours total
- Ran on an EtBr stained 3.8% agarose gel alongside GFP+B0024 ligation from 7/22 to see if ligation was successful (and the problem was with the colony we picked)
- GFPf+tt did not show a band ~800bp
- GFP ligation was okay the first time around (we picked a bad colony to plasmid prep?)
Reconstruction of assemblies
- Grace & Krystle
- PCR reaction to retrieve ccdB gene and BioBrick MCS from pSB1A3.
- Ran on gel to verify PCR product.
- Gel purified PCR product.
- RE digested overnight in 20 μl reactions (10μl DNA):
- pRL1383a with HindIII in NEBuffer 2
- ccdB+MCS with HindIII in NEBuffer 2
- GFP with EcoRI and SpeI in NEBuffer EcoRI + BSA
- GFP fusion with EcoRI and SpeI in NEBuffer EcoRI + BSA
- nir with EcoRI and SpeI in NEBuffer EcoRI + BSA
- rbs (B0030) with XbaI in NEBuffer 2 + BSA
- tt (B0024) with XbaI in NEBuffer 2 + BSA
Drylab Work
Primer Design
- Margaret
- I want to design primers for some replication proteins + origin from pSB2K3
Discussion
Quote of the Day
History is the only laboratory we have in which to test the consequences of thought. - Étienne Gilson
[http://manoa.hawaii.edu/ 
][http://manoa.hawaii.edu/ovcrge/ 
][http://www.ctahr.hawaii.edu 
]
