IGEM:MIT/2007/Notebook/2007-8-6
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Agenda
- Look at digestions on gel
- PCR Purify or Nucleotide Purify digestion results
- Ligate constitutive promoters to RBS, CPX to B0014
Expected Part Lengths
Name Part Length Plasmid Length I14032, P(Lac)IQ 37 bp 4425 bp (pSB1AK3) R0051, Lambda cl 49 bp 2079 bp (pSB1A3) I728500, CPX+poly. 720 bp 3500 bp (GeneArt standard vector) B0014, terminator 12 bp 3189 bp B0034, RBS 12 bp 2079 bp (pSB1A3)
Gel came out fine, but bands are faint.
Digestion Purification Results
Part Nanodrop I14032 4.7 ng/µL R0051 2.3 R0014 8.3 CPX 2.3 B0034 3.8
Ligation Mixtures
Also tested negative controls (same mixture, but no insert and additional water to equal volume)
I14032.B0034:
- 10 µL digested I14032
- 0.5 µL B0034
- 2 µL T4 Ligase buffer
- 0.5 µL T4 Ligase
- 7 µL H20
- 20 µL Total
R0051.B0034:
- 25 µL R0051
- 0.5 µL B0034
- 3 µL T4 Ligase Buffer
- 0.5 µL T4 Ligase
- 1 µL H20
- 30µL Total
CPX.B0014:
- 6 µL B0014
- 20 µL CPX
- 3 µL T4 Ligase Buffer
- 0.5 µL T3 Ligase
- 0.5 µL H20
- 30 µL Total