IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-8-11: Difference between revisions

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*<s>Digestion of GeneArt plasmids with X-P so they can be ligated immediately into J04500 (Lac + RBS) </s> (Dave)
*<s>Digestion of GeneArt plasmids with X-P so they can be ligated immediately into J04500 (Lac + RBS) </s> (Dave)
*Transformation/Plating of GeneArt plasmids
*Transformation/Plating of GeneArt plasmids
*Running digestion assay products + J04500 on a gel (Double H)
*<s>Running digestion assay products + J04500 on a gel </s> (Double H)
**Gel-purify a sh**load of things (GFP dev\X-P, pSB4A3\S-P, J04500\S-P)
**Gel-purify a sh**load of things (GFP dev\X-P, pSB4A3\S-P, J04500\S-P)
**Ligation assay (GFP dev\X-P + pSB4A3\X-P) along with J04500\S-P + KaiA\X-P, transformation
**Ligation assay (GFP dev\X-P + pSB4A3\X-P) along with J04500\S-P + KaiA\X-P, transformation
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Digested at 37&deg;C for 6 hrs, heat shock at 80&deg;C for 20 min, hold at 4&deg;C.
Digested at 37&deg;C for 6 hrs, heat shock at 80&deg;C for 20 min, hold at 4&deg;C.
Tubes labeled A,B,C respectively, with 8/11 on the side.
Tubes labeled A,B,C respectively, with 8/11 on the side.
==Digest Gels==
Two 1% agarose gels were made.
First agarose gel contains:
L (5 uL), L (5 uL), 1 (2h), 2 (2h), 3 (2h), 4(2h), 1 (4h), 2 (4h), 3 (4h), 4 (4h), 1 (6h), 2 (6h), 3 (6h), 4 (6h), L (10 uL)
Second agarose gel contains:
L (5 uL), 1 (12h), 2 (12h), 3 (12h), 4(12h), 1 (16h), 2 (16h), 3 (16h), 4 (16h), L (5 uL),  J04500 1 /S-P, L (10 uL)
The gels seem to be clogged up.  The bands are not running uniformly - there could be too much DNA in each of the wells.

Revision as of 10:36, 11 August 2006

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Ligation assay

We want to keep our ligation assay modest in size because each ligation requires at least a transformation (and possibly an inoculation and miniprep as well).

Ligation # Insert:Vector Ligation time
1 1:1 5 min
2 3:1 5 min
3 6:1 5 min
4 1:1 15 min
5 3:1 15 min
6 6:1 15 min
7 1:1 30 min
8 3:1 30 min
9 6:1 30 min

ToDo

  • Digestion of GeneArt plasmids with X-P so they can be ligated immediately into J04500 (Lac + RBS) (Dave)
  • Transformation/Plating of GeneArt plasmids
  • Running digestion assay products + J04500 on a gel (Double H)
    • Gel-purify a sh**load of things (GFP dev\X-P, pSB4A3\S-P, J04500\S-P)
    • Ligation assay (GFP dev\X-P + pSB4A3\X-P) along with J04500\S-P + KaiA\X-P, transformation
  • Prepare KaiA for sequencing
    • Gel-purify undigested KaiA + RFP bkb (H.C.) (Jeff)
    • Prepare sequencing order with BB primers (Jeff)
  • Perry's Pet Projects for Personal Persual (Peng)
    • Frozen stocks of all his stuff & innoculation
    • Colony PCR
    • Post-PCR gel (egel)

Digestion of GeneART plasmids

For each KaiA, kaiB, kaiC plasmid:

  • 4 uL DNA (~200 ng/uL gives 800ug DNA)
  • 17.25 uL H2O
  • 2.5 uL buffer (Buffer 3)
  • 0.5 uL enzyme 1 (XbaI)
  • 0.5 uL enzyme 2 (PstI)
  • 0.25 uL BSA

25 uL total volume.

MM:

  • 4x17.25= 69 dh20
  • 4x2.5= 10 buffer 3
  • 4x0.5 = 2 XbaI
  • 4x0.5 = 2 PstI
  • 4x0.25 = 1 BSA (be sure to mix well!)

pipet 21 into each tube containing the DNA


Digested at 37°C for 6 hrs, heat shock at 80°C for 20 min, hold at 4°C. Tubes labeled A,B,C respectively, with 8/11 on the side.

Digest Gels

Two 1% agarose gels were made.

First agarose gel contains:

L (5 uL), L (5 uL), 1 (2h), 2 (2h), 3 (2h), 4(2h), 1 (4h), 2 (4h), 3 (4h), 4 (4h), 1 (6h), 2 (6h), 3 (6h), 4 (6h), L (10 uL)

Second agarose gel contains:

L (5 uL), 1 (12h), 2 (12h), 3 (12h), 4(12h), 1 (16h), 2 (16h), 3 (16h), 4 (16h), L (5 uL), J04500 1 /S-P, L (10 uL)

The gels seem to be clogged up. The bands are not running uniformly - there could be too much DNA in each of the wells.