IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-8-28

Nick's request:
 * 8ul Midiprep Backbone DNA + .5 ul S+P enzyme + 2.5ul Buffer #2 + 2.5ul BSA + 10ul H2O-> 45C 6H
 * 8ul Midiprep KaiA DNA + 1 ul S+P enzyme + 2.5ul 10x Buffer #2 + 2.5ul 10x BSA + 10ul H2O-> 45C 6H
 * 8ul Midiprep KaiB DNA + 1 ul X+P enzyme + 2.5ul 10x Buffer #2 + 2.5ul 10x BSA + 10ul H2O-> 45C 6H
 * 8ul Midiprep KaiC DNA + 1 ul X+P enzyme + 2.5ul 10x Buffer #2 + 2.5ul 10x BSA + 10ul H2O-> 45C 6H

Digestion of BioBrick vector
Each KaiC:
 * 1) *4 uL DNA (42)
 * 2) *1 µL 10 x BSA (10.5)
 * 3) *1 µL 10x buffer #3 (10.5)
 * 4) *0.2 µL Xba1 (2.1)
 * 5) *0.2 µL Pst1 (2.1)
 * 6) *3.6 uL H20 (37.8)


 * 1) 10 samples
 * 2) Incubate for 4h@45

Each J04500:
 * 1) *4 uL DNA (22.4)
 * 2) *1 µL 10 x BSA (6.5)
 * 3) *1 µL 10x buffer #2 (6.5)
 * 4) *0.2 µL Spe1 (1.3)
 * 5) *0.2 µL Pst1 (1.3)
 * 6) *3.6 uL H20 (23.4)


 * 1) 6 samples
 * 2) Incubate for 4h@45

Then CIP treated J04500 w/ 1uL CIP for 1h

Running KaiC and J04500 on gel
This was the fun part; ran 10 KaiC and 6 J04500 on gel, but DID NOT use ETBR (found a paper which said it can reduce by 90% ligation efficiency when DNA w/ETBR exposed for 1min, and it interlocates which kills ligation efficiency). Used a "marker lane," ran gel, cut out the "marker lane" and post-stained in TBE+EtBr, UV imaged the marker lane and cut out band location, then matched it to the non-stained gel and cut corresponding area. Done for both; see below images.

Gel Purification AND PCR purification of KaiC and J04500
Did a gel purification of KaiC and J04500. Most importantly: 1) Definately do the extra QG wash 2) Let the DNA sit in PE for awhile (5min) 3) Did a third spin to get rid of excess PE 4) PreHeated EB to 70C 5) Eluted in 50 of EB

Note that eluting in H20 is uber-bad; the pH can be as low as ph5 out of the millicue. Nick also did experiments showing that 1/3EB + H20 is bad.

The extra PCR purification step was to combine all 6 J04500 and all 10 KaiC into one 30uL concentrated tube; this further cleans the DNA, concentrates it, and allows us to elute in EB (and not use the vacufuge). Got two colored tubes sitting in our freezer (30uL EB)

Miniprep and sequencing of Nick's 22.8 J04500 + KaiB transformant colonies
Last night we grew up 3 cultures inoculated from 3 colonies from Nick's 22.8 ligation (J04500 + KaiB). Colony #3 appeared to contain KaiB according to Nick's colony PCR. We miniprepped the cultures and sent all 3 off for sequencing.


 * CY100: 22.8 #1, VF2 primer
 * CY101: 22.8 #1, VR primer
 * CY200: 22.8 #2, VF2 primer
 * CY201: 22.8 #2, VR primer
 * CY300: 22.8 #3, VF2 primer
 * CY301: 22.8 #3, VR primer