Stanford/BIOE44:Module 5:Day3: Difference between revisions
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#Run uncut and single cut plasmids to see if there is even an intact plasmid. | #Run uncut and single cut plasmids to see if there is even an intact plasmid. | ||
#Try inoculating I0500 from different sources in case the source has been contaminated. | #Try inoculating I0500 from different sources in case the source has been contaminated. | ||
===Actions taken to fix I0500/pSB2k3 situation=== | |||
#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. | |||
#I also transformed [http://partsregistry.org/wiki/index.php?title=Part:BBa_K113009 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. | |||
#I inoculated K274111, K274120 and K274220 which are all on pSB2K3. | |||
Hopefully if everything goes well we will have pBAD and pSB2K3 for everyone! | |||
Latest revision as of 23:08, 25 May 2010
M5: Day 2 - Continue Building
Freezer Stocks of Synthesized Parts
Materials
- 5mL overnight culture
- sterile 60% glycerol
- 3 cryovials, labelled
Procedure
- Label your cryovials: part name, plasmid, date, your initials.
- Add 500ul of sterile 60% glycerol to each of your cryovials.
- 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)
- Invert tubes several times to mix.
- 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
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
- Run uncut and single cut plasmids to see if there is even an intact plasmid.
- Try inoculating I0500 from different sources in case the source has been contaminated.
Actions taken to fix I0500/pSB2k3 situation
- 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.
- 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.
- I inoculated K274111, K274120 and K274220 which are all on pSB2K3.
Hopefully if everything goes well we will have pBAD and pSB2K3 for everyone!
