IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-8-19

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To do tomorrow

 * 1. Miniprep of the J04500
 * 2: XP and SP digest of the above
 * 2. XP digest of the KaiA, B, and C from Geneart midiprepped source, enough for another ligation/transformation
 * 2. XP digest of the 13 miniprepped things below, only need an hour or two @45. On a red container in our fridge labeled "MINIPREP ME"
 * 3: e-gel of the 13 miniprepped things

To do monday

 * 1: If anything from the e-gel from above worked, then make glycerol stocks of it from the liquid culture in our fridge.
 * 1: CIP treatment of the newly digested J04500 backbone \sp and \xp
 * 2: Run the newly digested J04500\sp and \xp, the kai A, B, and C from above, AND the B0034\XP and \SP from yesterday on a gel and gel purify
 * 3: Ligation/Transformation #4, see ligation/Transformation #3 from Thursday for the protocol.

Innoculation of J04500
Innoculated J04500 from 8-10 Top10F stock in 4mL of KAN resistanace.

CIP of digest products
CIPPED the digest products, except I accidentally CIPed the KaiABC=(. We'll have to redigest those... and the backbone we have for J4500 is suspicious.

'''The post-CIP pre-gel purify B0034 \sp (7,8), J04500 \sp (9,10) and J04500 \xp (11,12) are in 0.5mL tubes labeeld with numbers on the top and a label on the side in our freezer. I would advise against using the J04500 however because I think that has J04450 contamination in it...'''--Zsun 21:07, 19 August 2006 (EDT)

Miniprep of innoculated cultures
I innoculated and miniprepped 13 things:


 * A: 4500\SP B from 8/16, ("1")
 * B: 4500\SP B from 8/16, ("2") (Note: could be swapped with 4500 \XP C #1)
 * C: 4500\SP C from 8/16, ("5")
 * D: 4500\SP B #1
 * E: 4500\XP C #2
 * F: 4500\SP B from 8/16, ("3")
 * G: 4500\SP B #2
 * H: 4500\XP A #1
 * I: 0034\SP A #1
 * J: 0034 \SP A #2 (Note: could be swapped with 4500\XP A #2)
 * K: 4500 \XP C #1 (Note: could be swapped with J04500\SP B from 8/16)
 * L: 0034 \SP C
 * M: 4500\XP A #2 (Note: could be swpped with 0034\SP A #2)

I know that looks confusing, but if someone could digest all of those with XP following the letter convention, we can run a diagnostic E-gel and see what is going on. For all of them, I HAVE NOT made glycerol stocks but they are all sitting in our fridge; do not toss! --Zsun 21:12, 19 August 2006 (EDT)

Red Mystery
Turns out one of the 4500 \XP C glows RED! This is just like the 4500\SP B sample (B) or (F) from the above table. Because 4500 \XP C was the only plate which had CARB resistance instead of KAN resistance, most likely our other plates also had colonies which glowed red. Possibilities:


 * mCHERRY: unlikely as the innoculation was done in two seperate places and two seperate experiments had the red color
 * the J4500 midiprepped sample: They all share this in common, so I suspect this is the sourse
 * the registry sample from where J4500 came from...

The easy way to solve this, I think, is to re-innoculate frozen cultures of J4500 in KAN resistance, which the contaminating J4450 does not have. --Zsun 21:12, 19 August 2006 (EDT)