Team:EPF-Lausanne/Notebook/October2011

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For the K1 backbone, the recipe was slightly different due to the higher concentration of blunted DNA we wanted to achieve:
For the K1 backbone, the recipe was slightly different due to the higher concentration of blunted DNA we wanted to achieve:
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* H20 - 4.4 uL
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* NEBuffer2 (10X, found in enzyme ice box) - 4 uL (needs to be at a final concentration of 1X)
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* dNTP (10 mM) - 0.4 (needs to be at a final concentration of 100 uM)
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* template - 30 uL (for 1.5 ug of DNA, assuming the T7 + Lysis was at a concentration of 100 ng/uL)
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* T4 Polymerase - 1.2 uL (it comes at a "concentration" of 3000 units/mL and we want 1.5 units)
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for a total of 40 uL.
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== Sunday, October 23 2011 ==
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Vincent ran a colony PCR on the 1 and 13 T7 promoter + Lysis + psB3K1 constructs, since they were the ones that yielded the highest colony count (compared to the negative control). He chose ten colonies from each plate. The protocol was as followed:
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* TPB 2.5 uL
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* EPFL Taq 0.25 uL
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* primer FW (10 uM) 0.5 uL (1289_F, originally 100 uM)
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* primer RV (10 uM) 0.5 uL (pSB-Pcon-TetR-r, originally 20 uM)
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* template 2 uL of DNA (boiled)
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* dNTP 0.5 uL
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* dH20 18.75 uL
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25 uL total
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The cycle (now called EPFLCOP for EPFL Colony PCR) is as follows:
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* 94 °C, 2 minutes
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30 times:
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* 94 °C, 15 sec.
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* 58 °C, 15 sec.
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* 68 °C, 1 minute (1 min / kb, and we expect 629 bp)
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End of 30
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* 68 °C, 5 min
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* 10 °C, infinitely
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The gel showed a strong signal at 630 bp for two colonies of T7-1 but none for T7-13. Unfortunately, this is the same promoter as the one amplified by Alina two months ago, so we can't use it to run an interesting experiment.
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[[File:Trashed t7promgel.jpg|500px]]
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== Monday, October 24 2011 ==
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Vincent mini-prepped the liquid culture from the successful T71-Lysis-K1 plasimd identified by PCR the previous day. He then transformed that DNA into BL21 cells and plated the transformant.
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Since the primers used on Sunday would miss half of the inserts (the blunt-end strategy implies that using primers on the insert and on the vector will miss half of the potentially good plasmids), we chose to switch to different primers: the 816_R and 30_F which amplify on the lysis cassette.
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With these primers, Vincent ran a colony PCR on 10 colonies per plate for the 2, 3, 4, and 6 plates (2,3,4, and 6 refers to the T7 promoter type). He used the same protocol as on Sunday. The resulting gel shows that 3 out of the 4 T7 variants show an amplification on some colony:
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[[File:2011-10-24-t7-ligation-colPCR-1.jpg|400px]]
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[[File:2011-10-24-t7-ligation-colPCR-2.jpg|400px]]
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== Tuesday, October 25 2011 ==
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Nadine and Douglas came in and mini-prepped liquid cultures prepared the previous day by Nadine.
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Vincent transformed the resulting DNA into BL21 cells.
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== Wednesday, October 25 2011 ==
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Late on Tuesday, Henrike noticed that the colonies had grown sufficiently to make liquid cultures for a platereader. Nadine ran the platereader on Wednesday with the following results:
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[[File:t7_c2_lysis_col1.png|400px]]
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[[File:t7_2_lysis_col7.png|400px]]
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[[File:t7_3_lysis_col3.png|400px]]
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[[File:t7_4_lysis_col8.png|400px]]
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Latest revision as of 06:54, 28 October 2011

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