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:

\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)



 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.



 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.