IGEM:IMPERIAL/2006/LabCalendar/2006-8-3

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)
 * 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.

Annealing Oligos

 * 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
 * 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