IGEM:IMPERIAL/2006/LabCalendar/2006-8-3: Difference between revisions
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*Propose to re-maxiprep all parts that were maxi-preped yesterday on 4/8 Friday | *Propose to re-maxiprep all parts that were maxi-preped yesterday on 4/8 Friday | ||
*PCR was also unsuccessful due to maxi-preping issues yesterday | *PCR was also unsuccessful due to maxi-preping issues yesterday | ||
==PCR fusion Protein== | |||
*Prepare on ice: Mix in a PCR tube the following: | |||
**28.5 ul pure water | |||
**5ul buffer (taq buffer containg sulphate) | |||
**1ul of each primer | |||
**10ul MgCl2 (from 25uM stock solution) | |||
**2.5ul DNTP mix (10uM) | |||
*Dilute 1ul of maxiprep DNA nto 50ul water. | |||
**Take 1ul of this solution and add to reaction mixture. | |||
**Add 1ul of Taq Polymerase | |||
*Total Volume should be 50ul. | |||
*Program PCR machine | |||
**94C for denaturation | |||
**Work out metling temperature from primer composition according to formula below. Also found on label of primer tube. | |||
==Set up Maxi-Prep for 4/8== | ==Set up Maxi-Prep for 4/8== |
Revision as of 06:24, 4 August 2006
Start Ligations
- Began stage 2 ligations
- (12D->24A)->6B
- F2620 (6B) - Vector (Cut with S & P, use orange buffer)
- C0261+I13504 (12D->24A) - Insert (Cut with X & P, use yellow buffer)
- (7A->3O)->9G
- R0062 (9G) - Vector (Cut with S & P, use orange buffer)
- B0034+C0062 (7A->3O) - Insert (Cut with X & P, use yellow buffer)
- (12D->24A)->6B
- Digests and gel running unsuccessful
- No DNA was evident on the gel, thought to have been caused by incorrect maxi-prep of parts
- Propose to re-maxiprep all parts that were maxi-preped yesterday on 4/8 Friday
- PCR was also unsuccessful due to maxi-preping issues yesterday
PCR fusion Protein
- Prepare on ice: Mix in a PCR tube the following:
- 28.5 ul pure water
- 5ul buffer (taq buffer containg sulphate)
- 1ul of each primer
- 10ul MgCl2 (from 25uM stock solution)
- 2.5ul DNTP mix (10uM)
- Dilute 1ul of maxiprep DNA nto 50ul water.
- Take 1ul of this solution and add to reaction mixture.
- Add 1ul of Taq Polymerase
- Total Volume should be 50ul.
- Program PCR machine
- 94C for denaturation
- Work out metling temperature from primer composition according to formula below. Also found on label of primer tube.
Set up Maxi-Prep for 4/8
Cultured the following in 50 ug/mL Amp LB
- 1I
- 5M
- 12D->24A colonies 1, 3, and 4
- 16P
- 7A->3O colony 2
- 18I (electroporation successful)
Part I13207 Testing
- Made up 10% L-arabinose solution
- 1 g into 1 mL MilliQ
- Made up 1M Zinc chloride solution
- 1.363 g in 10 mL MilliQ
- Protocol underway
- Will be tested in 2 phases
- Part 1 testing: Same as T9002 testing, can be done in parallel
- Part 2 testing: To see on or off of AiiA with key parameters (arabinose, zinc chloride)
To Do on Friday
- Maxiprep - 8:30 am
- John C, Jimmy, Christin, Tom
- Ligations - 10:30 am (dependent upon Maxiprep finishing)
- John S, Jonny
- PCR - 10:30 am (dependent upon Maxiprep finishing)
- Deepti, Farah, Christin
- Gel - 10:30 am (overnight PCR and maxipreps to check)
- A few people who do maxiprep