Stanford/BIOE44:Module 5:Day3

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M5: Day 2 - Continue Building

Freezer Stocks of Synthesized Parts

Materials

  • 5mL overnight culture
  • sterile 60% glycerol
  • 3 cryovials, labelled

Procedure

  1. Label your cryovials: part name, plasmid, date, your initials.
  2. Add 500ul of sterile 60% glycerol to each of your cryovials.
  3. Add 1.5mL of overnight culture to each cryovial. (Its easiest to use a 5ml pipette, take up 4.5mL of culture then drop 1.5mL into each tube)
  4. Invert tubes several times to mix.
  5. Put in freezer box in -80C freezer. (Usually one tube is kept as the working stock and the other two are kept as back ups in a separate freezer.)

PCR amplifying your parts

Your primers have arrived! This is the packing list that comes with the primers when they are delievered. It has a lot of useful information.

We will use this polymerase: Pfx50 DNA polyermase It is higher fidelity than TAQ (which means it makes less mistakes).

PCR mix (taken from Pfx50 manual)

Component Volume Final Conc.
10X Pfx50 buffer 5ul 1X
10mM dNTPs 1.5ul 300uM
fwd primer 300nM
rev primer 300nM
Template 1-100pg
Pfx50 polymerase 1ul
Water
Total volume 50ul


PCR Protocol (taken from Pfx50 manual)

Cycles Temp Time
1x 94C 2 min
35x 94C 15s
60-68C (Tm primers - 2C) 30s
68C 30s per kb
1x 68C 5 min
1x 15C forever

I0500 troubleshooting

I0500 gel

When I0500 (in pSB2K3) is digested with EcoRI & PstI a large smear is produced. When the same construct is digested with SpeI & PstI, a smear, albeit smaller, is also produced.

Smears are caused by a whole bunch of pieces of DNA that are only slightly different in size. This can be caused by degradation of DNA products. Multiple people have had this happen when they digested this vector. What could be happening? Why would digestion with different enzymes change the smear?

Troubleshooting ideas

  1. Run uncut and single cut plasmids to see if there is even an intact plasmid.
  2. Try inoculating I0500 from different sources in case the source has been contaminated.

Actions taken to fix I0500/pSB2k3 situation

  1. I found a second Spring 2009 Plate 1 (not sure where this came from, I0500 had been taken from first Plate 1 I found) but I transformed I0500 from this plate.
  2. I also transformed BBa_K113009 which is also the AraC/pBAD promoter. It was build by the iHKU iGEM team because they were unhappy with I0500. It is on pSB1A2.
  3. I inoculated K274111, K274120 and K274220 which are all on pSB2K3.
Hopefully if everything goes well we will have pBAD and pSB2K3 for everyone!
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