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°C for 6 hrs, heat shock at 80°C for 20 min, hold at 4°C. | 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. | 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.