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Team:British Columbia/Notebook/Week 5
From 2011.igem.org
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Lab Meeting - July 3
The team started tracking the cost of flights to Indianapolis for the Regionals; in addition, they began working on the project proposal and safety page that is due next week!
3-Carene
Daisy has been trying to figure out why the sequencing did not work. It may be due to the miniprep method. She will continue to make the yeast construct and if the sites are still there, she can always do another mutagenesis on the yeast plasmid.
1,8-Cineole
Jacob ran a restriction digest and gel to check his variant SDM-PCR product. It was successful! However, his attempt to remove another restriction site from pg-tps-cin did not work, so Jacob ran another SDM with more PFU - it worked! Jacob then transformed E. coli with this product. Using plasmid mini-prepped from successful transformants, Jacob proceeded to SDM the next restriction site...
... which also worked! Now, both restriction sites from pgxe-tps-cin and 2 of 3 restriction sites from pg-tps-cin have been successfully removed. One more to go! Jacob set up the (hopefully) final SDM to run overnight...
Success! The gel showed that the SDM was successful and another gel, which showed a restriction digest of the previous SDM products suggests that the SDMs were all successful. Jacob then did the final transformation with the fully SDMed products.
beta-Pinene & (-)-Limonene
Marianne repeated her SDM of beta-pinene by using both Pfu and Taq. Gel verification showed a smear so she decided to run a gel on the mini-prep of the beta-pinene plasmid from Rafael to make sure that the DNA wasn't contaminated... Fortunately, there were bands at the correct sizes indicating that the plasmid is fine. So she repeated the SDM using temperature-ramping...
Finally! Gel verification of the SDM beta-pinene product shows bands of the correct size suggesting that the SDM was successful. This was transformed into E. coli.
However, the SDM limonene product appeared as smears and various bands, suggesting unspecified annealing of primers. More troubleshooting: DMSO could be lowering the Tm of the primers; repeat without adding DMSO for one sample, without adding MgCl2 for another sample, use the ramping cycling condition previously indicated. However, even after these modifications, further SDMs did not yield the desired product... Vicki is very frustrated!
Extra Parts: IDI1, HMG2, erg20-2
We received and amplified 5 plasmids from Greece containing various parts and synthase genes: IDI1, HMG2, p300, Cin, Sab. These plasmids were transformed into yeast to make sure they were in working order.
Still awaiting the erg20-2 part from France...
alpha-Pinene
After some very useful advice from the graduate advisor, Joe speculates that secondary structures or primer-dimer may be the reason why he hasn't gotten any PCR products containing his SDMed- alpha pinene synthase. This time, he will add additional DMSO to remove any formation of secondary structures. Hopefully this will allow his PCR reaction to be successful.
He found an optimal Quikchange PCR reaction mixture (for 50uL reaction):
| Reagents | Volume |
|---|---|
| ddH2O | 37.5uL |
| 10X Rxn Buffer | 5uL |
| dNTP (25mM | 1uL |
| Ps-TPS-Pin Fw(125ng) | 1uL |
| Ps-TPS-Pin Re(125ng) | 1uL |
| Pfu polymerase | 1uL |
| DNA template (50ng/uL) | 1uL |
| DMSO | 2.5uL |
Cycling Conditions:
| Temperature | Time | Cycles |
| 95oC | 30 sec | 1 |
| 95oC | 1 min | 16 |
| 55oC | 1 min | |
| 68oC | 10 min | |
| 4oC | Hold | |
He did a gel electrophoresis on his SDM-PCR products while he DpNI digested his product for 1 hour at 37C. The gel shows a product at 7-8kb. Again, he can't conclude anything until he does a gel electrophoresis on his DpnI digested products. After waiting for 1 hour, he finally did a gel electrophoresis on his DpnI digested products. A thin but more distinct band was seen on the gel and it had the same molecular weight as his earlier gel (showing his PCR products). Further, at the bottom of the gel and beyond his ladder is a "glow" of bands, which may be the digested products of his template or primer-dimer He is now certain that his SDM PCR worked and that the DpnI digested all his template away, leaving SDM-PCR products left in the PCR tubes. He is ready for the transformation. Horray!
Joe transformed his DpnI digested products into DH5-alpha cells and plated them on kanamcyin plates. He transformed with 5uL, 10uL, 20uL of DpnI digested products, hoping that one of them will give him colonies. In the end, only 3 colonies grew on the plate containing transformants created using 20uL of the DpnI digested products. Joe then cultured all three colonies and performed a mini-prep to purify and isolate the desired SDMed-alpha pinene synthase.
