IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-8-13: Difference between revisions

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==Ligation of GeneArt Plasmids, natural KaiA into J04500 backbone ==
==Gel purification, Ligation and transformation of GeneArt Plasmids, natural KaiA into J04500 backbone ==


I gel purified the inserts pre-ligation, but the nanodrop gave extremely low (eg. negative) numbers. Then, I used an absolute ratio of 1:3 vector-insert instead of the normal molar ratio. DO NOT USE THIS PROTOCOL FOR SUBSEQUENT LIGATIONS!


For each kaiA, kaiB, and kaiC plasmid:
'''Ligation for samples the nanodrop hates'''<br>
*BB vector J04500 \SP: 2.5uL
*BB insert KaiA, B, or C \XP: 7.5uL
--------


* 4 uL DNA (~200 ng/uL gives 800ug DNA)
Protocol:
* 17.25 uL H<sub>2</sub>O
*Mix above ratios + 10uL #1 + 1uL #3
* 2.5 uL buffer (Buffer 3)
**BE sure to mix #1 (ligation buffer) well especially!
* 0.5 uL enzyme 1 (XbaI)
*Sit at bench for ~30min
* 0.5 uL enzyme 2 (PstI)
*Put back on ice
* 0.25 uL BSA


25 uL total volume.
'''Transformation'''


MM:
For the transformation I did 5 samples:
*4x17.25= 69 dh<sub>2</sub>0
*1 exp. (50uL) natural KaiA
*4x2.5= 10 buffer 3
*1 exp. (50uL) synthetic KaiA
*4x0.5 = 2 XbaI
*1 exp. (50uL) synthetic KaiB
*4x0.5 = 2 PstI
*1 exp. (50uL) synthetic KaiC
*4x0.25 = 1 BSA (be sure to mix well!)
*1 (25uL) positive
*1 (25uL) negative
 
Ice incubation 30m, 30s@42C, 2m@ice, SOC@1h@37C. Plated on LBCarb. Should be ready around 6PM to see.

Revision as of 10:02, 13 August 2006

Gel purification, Ligation and transformation of GeneArt Plasmids, natural KaiA into J04500 backbone

I gel purified the inserts pre-ligation, but the nanodrop gave extremely low (eg. negative) numbers. Then, I used an absolute ratio of 1:3 vector-insert instead of the normal molar ratio. DO NOT USE THIS PROTOCOL FOR SUBSEQUENT LIGATIONS!

Ligation for samples the nanodrop hates

  • BB vector J04500 \SP: 2.5uL
  • BB insert KaiA, B, or C \XP: 7.5uL

Protocol:

  • Mix above ratios + 10uL #1 + 1uL #3
    • BE sure to mix #1 (ligation buffer) well especially!
  • Sit at bench for ~30min
  • Put back on ice

Transformation

For the transformation I did 5 samples:

  • 1 exp. (50uL) natural KaiA
  • 1 exp. (50uL) synthetic KaiA
  • 1 exp. (50uL) synthetic KaiB
  • 1 exp. (50uL) synthetic KaiC
  • 1 (25uL) positive
  • 1 (25uL) negative

Ice incubation 30m, 30s@42C, 2m@ice, SOC@1h@37C. Plated on LBCarb. Should be ready around 6PM to see.

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