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

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Current revision (11:00, 16 August 2007) (view source)
(Transformed Ligation of F+B+CPX)
 
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* 30 µL Total
* 30 µL Total
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==Transformed Ligation of F+B+CPX==
 
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*Used Joey's B-R e.coli cells (electrocompetent)
 
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Electrocompetent procedure:
 
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1. After ligation/denaturation, use pore to filter DNA (10µl) in water in petri dish
 
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2. Add DNA to 50µl of cells
 
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3. Add mixture to cuvette
 
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4. On machine, set to EC20(for e.coli) and measurement to time in ms
 
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5. Load cuvette with knob on right, ensuring that all fluid is on bottom
 
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6. Insert cuvette so that both metal electrodes are touching metal sides of cuvette
 
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7. Press Pulse button
 
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8. Time measurement should be approximately 6 ms (ours were around 5-5.3)
 
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9. Immediately add 1mL of LB to cuvette and transfer mixture back into eppendorf with LB
 
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10. For growing cells on Chloremphenicol media, let cells recuperate for 1 hour. This step is not necessary for Ampicillin.
 
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*Plate all 5 samples (cPCR6, cPCR8, -cPCR6, -cPCR8, F+B+CPX insert only) on both Chloremphenicol and Kanamyacin plates for antibiotic screening. Hopefully colonies will grow only on Chloremphenicol plates.
 
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*Plates placed in 37C at around 12am
 
==Notes on Future Assays==
==Notes on Future Assays==

Current revision

Contents

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.
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