IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-8-11: Difference between revisions
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0.25 uL BSA | 0.25 uL BSA | ||
===pSB4A3=== | ===pSB4A3 (undigested)=== | ||
19.5 uL pSB4A3 | 19.5 uL pSB4A3 | ||
2.75 uL H<sub>2</sub>O | 2.75 uL H<sub>2</sub>O | ||
2.5 uL Buffer 2 | 2.5 uL Buffer 2 | ||
0.25 uL BSA | 0.25 uL BSA | ||
Revision as of 15:38, 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:
- R0010+E0241 is in psB1A2 (2079bp); R0010+E0241 cut with XP is 200bp+795=995bp+6(mixed)+16+6(sides)=~1023kb
- Then, cut with X&P should be ~2.1kb and 1023bp; togther should be around 3-3.1kb
- psB4a3=3339bp (SP cut makes linear)
- psB4K3+J04500=3409bp
Results: Looks as if overall:
- the digest depends on the amount of enzyme used. 0.5uL enzyme was always better 0.5uL
- prehaps the digest is dependant on the PCR machine used
- The best digests for R0010, in order w/regards to the insert (1kb, is):
- 12h, 0.5uL ea.
- 12h, 1.0uL ea.
- 2h, 0.5uL ea.
- 16h, 0.5uL ea.
- The above results are a bit inconclusive; either 37C in a water bath over a PCR is better, or the digest is just finniky.
What this tells us:
- Always use 0.5uL ea. enzyme for double digest
- Use 12h digests if you have time; otherwise, 2h in a water bath seems to work, prehaps around 40C.
Redigest of pSB4A3\S-P, and pSB4A3\X-P
The pSB4A3 (low-copy plasmid) didn't digest quite right. It appears to be 4kb long instead of the expected 3.3kb. We will redigest it, along with an X-P digestion to look for an insert. This time we'll also run it alongside undigested plasmid for comparison.
pSB4A3\S-P
19.5 uL pSB4A3 1.75 uL H2O 2.5 uL Buffer 2 0.5 uL SpeI 0.5 uL PstI 0.25 uL BSA
pSB4A3\X-P
19.5 uL pSB4A3 1.75 uL H2O 2.5 uL Buffer 3 0.5 uL XbaI 0.5 uL PstI 0.25 uL BSA
pSB4A3 (undigested)
19.5 uL pSB4A3 2.75 uL H2O 2.5 uL Buffer 2 0.25 uL BSA
