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)
   • 1 µL 10 x BSA (10.5)
   • 1 µL 10x buffer #3 (10.5)
   • 0.2 µL Xba1 (2.1)
   • 0.2 µL Pst1 (2.1)
   • 3.6 uL H20 (37.8)

1. 10 samples
2. Incubate for 4h@45

Each J04500:

1. • 4 uL DNA (22.4)
   • 1 µL 10 x BSA (6.5)
   • 1 µL 10x buffer #2 (6.5)
   • 0.2 µL Spe1 (1.3)
   • 0.2 µL Pst1 (1.3)
   • 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)

Nanodrop values obtained for pink and green vials (J04500/sp and KaiC /xp w/o ETBR)

KaiC

J04500 diluted 2x

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