IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-8-25

Miniprep of Nick's cultures #5 and #8

I miniprepped the 4 cultures Nick inoculated last night, and labeled them 5-1, 5-2, 8-1, and 8-2.

From Nick's email:

All three colonies are KaiB constructs. I have made 4 overnight cultures with the following labels:

"NES 5" is a putative KaiB insert in a J04500 from a 6:1 Insert:Backbone reaction using the NEB ligase. "NES 5" is a second culture started from the same colony. "NES 8" is a putative KaiB insert in a J04500 from a 3:1 Insert:Backbone reaction using the Roche ligase. "NES 8" is from a different (*not* clonal) colony from the same ligation reaction as the other NES 8.

Diagnostic digests of Nick's cultures #5 and #8

Digest #	Plasmid	Enzymes
1	5-1	X
2	5-1	X, P
3	8-1	X
4	8-1	X, P
5	8-2	X
6	8-2	X, P

\X digest reactions

• 19.5 µL DNA
• 2.25 µL H₂O
• 2.5 µL NEBuffer 2
• 0.5 µL XbaI
• 0.25 µL 100x BSA

\X digest master

• 9 µL H₂O
• 10 µL NEBuffer 2
• 2 µL XbaI
• 1 µL 100x BSA

Pipette 5.5 µL into each reaction.

\X-P digest reactions

• 19.5 µL DNA
• 1.75 µL H₂O
• 2.5 µL NEBuffer 3
• 0.5 µL XbaI
• 0.5 µL PstI
• 0.25 µL 100x BSA

\X-P digest master

• 7 µL H₂O
• 10 µL NEBuffer 3
• 2 µL XbaI
• 2 µL PstI
• 1 µL 100x BSA

Pipette 5.5 µL into each reaction.

Digested for 6 hours at 45C, set to finish around 2300 tonight.

Restreak and colony PCR

I restreaked, colony PCR'd, and inoculated 10 colonies from Nick's ligation #5 that he plated on 2006-8-24 (bottom of link). They are restreaked on two plates.

Ligation conditions:

KaiC Gel + Gel Backbone #5 II at 17C

Colony PCR reaction

• 8 uL PCR supermix
• 1 uL VF2 primer
• 1 uL VR primer

Colony PCR master mix

• 88 uL PCR supermix
• 11 uL VF2 primer
• 11 uL VR primer

Pipette 10 uL into each reaction.

Colony PCR schedule

• 95C for 15'00
• Do 30 times:
  • 95C for 0'30
  • 55C for 0'30
  • 72C for 2'00
• 72C for 10'00
• 4C forever

E-gel image of colony PCRs

It looks like the colonies don't contain our plasmid. (How can you tell? - See Below)

Click for legend

If there were no insert, we'd expect a 200bp band from a PCR. If there was no plasmid at all, our colonies would not have grown up under AMP selection. Ambiguously, no bands at all were seen.

E-gel image of \X and \X-P digests of Nick's KaiB + J04500 ligations

I forgot to run the undigested plasmid on this gel, but by comparing the \X and \X-P digests, it appears we still don't have insert. (How can you tell? - See Below) We expect ~530bp insert from this ligation.

Click for legend

I'm having trouble interpreting this gel. The X-P cut should produce either a 530bp KaiB insert, a 200bp lac-rbs insert, or no insert at all. None of these options appear; we appear to get multiple 3+kb bands. Also, singly cut plasmid produces two bands, whereas doubly-cut plasmid produces only one band. It seems unclear how the presence or absence of any insert can be determined from this gel.