iGEM 2013 — Team Waterloo

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# Team:Waterloo/Notebook

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| [Home](/Team%3AWaterloo "Team:Waterloo") | [Team](/Team%3AWaterloo/Team "Team:Waterloo/Team") | [Official Team Profile](https://igem.org/Team.cgi?year=2013&team_name=Waterloo) | [Project](/Team%3AWaterloo/Project "Team:Waterloo/Project") | [Parts Submitted to the Registry](/Team%3AWaterloo/Parts "Team:Waterloo/Parts") | [Modeling](/Team%3AWaterloo/Modeling "Team:Waterloo/Modeling") | **Notebook** | [Safety](/Team%3AWaterloo/Safety "Team:Waterloo/Safety") | [Attributions](/Team%3AWaterloo/Attributions "Team:Waterloo/Attributions") |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |

March 13th, 2013: Made stocks of 6 oligos which will be annealed to make the PhiC31 attP site.

March 18th, 2013: Inoculated strain #118 from frozen stock.

March 19th, 2013: The cultures from yesterday did not grow. Streak out strain #118 onto regular LB and Cm + LB to check for contamination of stock.

March 20th, 2013: There were multiple morphologies of colonies on the Cm + LB plate from yesterday therefore the frozen stock is likely contaminated.

March 21st, 2013: Make new frozen stock of DH5-alpha carrying pSB1C3 + RFP stuffer fragment (strain #151).

March 22nd, 2013: Maxiprep pSB1C3 from strain #151.

March 26th, 2013: Restriction digest of pSB1C3 with EcoRI and PstI showed a smear on the gel.

March 27th, 2013: Restriction digest of pSB1C3 from maxi-prep showed genomic DNA contamination. We were able to gel extract pSB1C3 from the gel to separate it from genomic DNA contamination. Transform DH5-alpha with gel extracted pSB1C3.

March 28th, 2013: Transform plate showed red colonies. Our competent cells work.

April 1st, 2013: Inoculated strain #151.

April 2nd, 2013: Hpdo is received as miniprep and agar stab. Streak out Hpdo on a LB + Km plate. Miniprep pSB1C3. Nanodrop received Hpdo but we got a bad concentration maybe because we do not know what buffer to blank with. The Hpdo streak plate grew. Store Hpdo streak plate in the fridge.

April 3rd, 2013: Restriction digest of pSB1C3. Gel extract the linearized pSB1C3 cut with EcoRI and PstI. The gel extracted samples did not contain DNA so we discarded them. We are not sure what went wrong. Someone forgot to take a picture of the gel. Inoculate strain #151.

April 4th, 2013: Miniprep pSB1C3.

April 5th, 2013: Maxiprep Hpdo. This later turned out to be contaminated with genomic DNA.

April 6th, 2013: Restriction digest of pSB1C3 shows genomic DNA contamination. Inoculate strain #151.

April 7th, 2013: Miniprep pSB1C3 from strain #151.

April 8th, 2013: Restriction digest of pSB1C3 with EcoRI and PstI. Gel extract linearized pSB1C3.

April 10th, 2013: Ligation of 6 oligos to form the PhiC31 attP site into pSB1C3.

April 11th, 2013: Diagnostic gel to check linearized pSB1C3 from April 10th, 2013. No gel red added no bands observed.

April 12th, 2013: Transformation of ligated product from April 10th, 2013. No colonies were seen. At this point we are not sure why.

April 16th, 2013: Restriction digest of pSB1C3 with EcoRI and PstI. Gel extract linearlized pSB1C3.

April 17th, 2013: Diagnostic gel confirms gel extracted product from yesterday is linear and of the expected size.

April 22nd, 2013: Ligation of 6 oligos to form the PhiC31 attP site into pSB1C3.

April 23rd, 2013: Transformation of ligated product from April 22nd, 2013. No colonies were seen. At this point we are not sure why.

April 24th, 2013: Transformation control using pSB1C3 confirms our competent DH5-alpha cells are still good.

April 30th, 2013: Ligation to check ligase activity.

April 31st, 2013: Transformation to check ligase activity. Inoculate culture of strain #152 containing Hpdo.

May 1st, 2013: Miniprep Hpdo. Diagnostic gel confirms ligase is working correctly.

May 6th, 2013: Making Amp + LB plates and streaking out E. coli carrying pSH62. pSH62 has the gene encoding PhiC31 integrase.

May 7th, 2013: Streak plate from yesterday looks good, store in fridge. Inoculate cultures from that streak plate. Diagnostic gel from gel extracted samples done on April 17th, 2013 show linearized pSB1C3 even though nanodrop values are negitive. Inoculate cultures of strain #151 and streak out a fresh plate of strain #151.

May 8th, 2013: Inoculation from streak plate of E. coli carrying pSH62 did not grow. Inoculate again from the streak plate with fresh Terrific broth. Miniprep strain #151. Inoculate strain #152.

May 9th, 2013: Make frozen stock of DH5-alpha carrying pSH62 --> strain #153 and store in iGEM strain box 3. Miniprep strains #152 and #153. Restriction digest of pSB1C3 with EcoRI and PstI. The pSB1C3 looks good on the gel. Gel extract the samples. The nanodrop values of the gel extracted samples were bad.

May 10th, 2013: Run a diagnostic gel with our gel extracted samples from yesterday. Though the nanodrop values are bad the linearlized backbone is clearly visible at the expected size on a diagnostic gel. To find an estimate of the concentrations we ran the gel extracted samples from yesterday on an agarose gel next to 100ng of lambda / HindIII ladder. This indicated the gel extracted samples had a concentration of ~ 5ng/uL.

May 12th, 2013: Inoculate strains #154 and #155 containing pUC118 and pUC119.

May 13th, 2013: Make frozen stock of strains #154 and #155. Miniprep strains #154 and #155. Amplify Hpdo minus gene VIII with KOD from maxiprep, Standford gift sample, May 1st miniprep and May 9th miniprep.

May 14th, 2013: Gel extraction of Hpdo minus gene VIII. Run the gel extracted samples by a lambda / HindIII ladder to estimate the concentration.

May 15th, 2013: Concentrate pSB1C3 cut with EcoRI and PstI with the speed vac. Self ligation of Hpdo minus gene VIII. Amplify PhiC31 integrase for pSH62.

May 16th, 2013:

You should make use of the calendar feature on the wiki and start a lab notebook. This may be looked at by the judges to see how your work progressed throughout the summer. It is a very useful organizational tool as well.

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