Team:British Columbia/Notebook/Week 5

From 2011.igem.org

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'''Extra Parts: IDI1, HMG2, erg20-2'''
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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.
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Still awaiting the erg20-2 part from France...
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==July 04 2011==
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'''1,8-Cineole'''
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<b>IDI1, HMG2, erg20-2</b>
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*Validated competent cells by transforming with pet100 vector containing Cineole synthase.  They are in working order.
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*Amplified 5 plasmids received from Greece (IDI1, HMG2, p300, Cin, Sab).  NOTE: did not properly centrifuge during DNA precipitation stage.  Will validate plasmids in yeast transformations to make sure plasmids are in working order.
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*Awaiting materials from France (erg20-2).
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<p><b>1,8-Cineole</b></p>
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*Ran a gel to test restriction digest on variant SDM-PCR product. Looks like the SDM was successful!
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<p><b>beta-pinene & (-)-limonene</b></p>
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*SDM beta-pinene again by using both Pfu and Taq.
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*Gel verification shows a smear.
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*Troubleshooting: run a gel on the miniprep of the beta-pinene plasmid received from Rafael to make sure that the DNA hasn't been contaminated/digested.
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==July 05 2011==
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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 <i>E. coli</i> with this product. Using plasmid mini-prepped from successful transformants, Jacob proceeded to SDM the next restriction site...  
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<b>beta-pinene & (-)-limonene</b>
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*Ran a gel on the miniprep of beta-pinene and (-)-limonene (SDM template).
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*Shows bands at the correct sizes indicating that the plasmid is present.
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*SDM beta-pinene and (-)-limonene again using ramping (ramping rate of 0.2 degrees C/sec).
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... 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...  
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<b>1,8 Cineole</b>
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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.  
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*Jacob tried to remove another restriction site from pg-tps-cin. The SDM did not work.
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==July 06 2011==
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'''Beta-pinene & (-)-limonene'''
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<b>beta-pinene</b>
+
-
*Gel verification of the SDM product shows bands of the correct size suggesting that the SDM was successful.
+
-
<p><b>(-)-limonene</b></p>
+
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...  
-
*Gel verification of the SDM product shows some lanes with smears, and another with 2 bands suggesting unspecified annealing of primers. SDM failed.
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*Troubleshooting: DMSO could be lowering the Tm of the primers; Repeat the SDM PCR without adding DMSO for one sample, without adding MgCl<sub>2</sub> for another sample. Use the ramping cycling condition previously indicated.
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**Gel verification of the SDM product shows no bands. Addition of DMSO and MgCl<sub>2</sub> is not the problem.
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<b>1,8-Cineole</b>
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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 <i>E. coli</i>.
-
*Ran another SDM on pg-tps-cin, this time with more PFU. The gel suggests that it worked! Jacob then transformed E. coli with this product.
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==July 07 2011==
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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 MgCl<sub>2</sub> for another sample, use the ramping cycling condition previously indicated. However, even after these modifications, further SDMs did not yield the desired product...
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<b>beta-pinene</b>
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*Transformed the SDM products.
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<b>1,8-cineole</b>
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*Colonies! The product from the SDM to remove a restriction site from pgxe-tps-cin successfully transformed E. coli. On to the next restriction site!
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==July 08 2011==
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<b>1,8-cineole</b>
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-
 
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Both restriction sites from pgxe-tps-cin and two of the three restriction sites from pg-tps-cin have been successfully removed using SDM. One more to go! Jacob set up the (hopefully) final SDM to run overnight.
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+
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==July 09 2011==
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'''1,8-cineole'''
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-
 
+
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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 are working. Jacob then did the final transformation with the fully SDM'd products.  
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'''Ampicillin Plates'''
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The team was running low on ampicillin plates, so Jacob made more and will test them soon.
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Revision as of 03:23, 4 August 2011

Team: British Columbia - 2011.igem.org

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...

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...

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