IGEM:MIT/2007/Notebook/2007-8-6

Agenda

1. Look at digestions on gel
2. PCR Purify or Nucleotide Purify digestion results
3. 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

Notes on Future Assays

• Try immunoassay with M9 media instead of LB. This should reduce background fluorescence
• Use a loading control, such as BSA, in next Western Blot to ensure fluorescence gradients across various levels of induction are not due to human error in loading. By adding equal amounts of BSA into all samples (assuming all samples are at equal volumes), the band for BSA(with a known molecular weight) should be of equal darkness across all samples.
• To test if the extra 4 bands we are getting on the Western Blot are due to expression of other proteins from our pSB1AC3 plasmid (such as those for amp or cmp. resistance), transform cells with plasmid without insert (perhaps just promoter + RBS without CPX) instead and compare with untransformed and transformed CPX cells.