- Began stage 2 ligations
- F2620 (6B) - Vector (Cut with S & P, use orange buffer)
- C0261+I13504 (12D->24A) - Insert (Cut with X & P, use yellow buffer)
- R0062 (9G) - Vector (Cut with S & P, use orange buffer)
- B0034+C0062 (7A->3O) - Insert (Cut with X & P, use yellow buffer)
- 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 (1min)
- Work out melting temperature from primer composition according to formula below. Also found on label of primer tube.
- (2xA-T)plus(4xG-C)-5= Melting temperature.(1 min)
- Calculate for both primers and use the lower of the two.
- 72C for extension (1min-1min 15 sec depending on template size. 1min for 1KB template).
- Run for 25 cycles
- Leave at 4C after cycles (program machine).
- Run DNA on gels to isolate.
- Oligos come as dry powder.
- Resuspend in water (add 1ul water for every ug of powder)
- Caution: careful when adding water - powdered disc may float out!
- In a PCR tube:
- Add 5ul each of the two complementary oligos
- Add 2ul of O-buffer (orange top)
- Make up the rest of the solution to 20ul with water
- Place epindorf in PCR machine for 2-3 mins at 94C
- Switch off machine and allow to cool for ~1hr to room temperature (oligos should anneal)
- To add phosphates to the strands:
- Mix 10ul of annealed oligos
- 2ul of 10 times Buffer A
- 1ul 10mM ATP
- 6ul dH20
- 1ul PNK
- Leave in 37C waterbath for 30-60mins
Set up Maxi-Prep for 4/8
Cultured the following in 50 ug/mL Amp LB
- 12D->24A colonies 1, 3, and 4
- 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