IGEM:Paris Liliane Bettencourt/Notebook/Paris 2010/2010/07/20

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Raphaël, Aleksandra and Théotime Quantification of DNA with the Nanodrop Samples from 07/19 prepared from the 07/17 miniprep Sample name 260/280 260/230 ng/µL attC mini 2.06 2.23 54.8 attC restr. 0.64 0.30 16.1 attC gel extr. 0.96 -0.01 -1.1 term-attC-mRFP restr. 0.78 0.41 17.5 term-attC-mRFP gel extr. 1.39 -0.05 -3.1 pLux (R0062) mini 1.88 2.19 101.0 pLux (R0062) restr. 0.83 0.46 22.3 pLux (R0062) gel extr. 4.56 -0.01 -0.5 RBS/GFP/term (E0240) restr. 0.93 0.67 23.1 RBS/GFP/term (E0240) gel extr. 1.79 0.05 7.0* Pstrong (J23119) restr. 0.79 0.43 19.7 Pstrong (J23119) gel extr. 0.10 0.00 0.0 Pmedium (J23110) restr. 1.26 0.87 49.3 Pmedium (J23110) gel extr. 1.63 0.05 23.7* RBS/LuxR (J37033) restr. 0.91 0.61 21.6 RBS/LuxR (J37033)gel extr. 2.03 0.01 3.8 RBS/LuxI (K081008) restr. 0.85 0.41 25.6 RBS/LuxI (K081008) gel extr. 0.60 0.00 0.3 term. (B0015) restr. 1.14 0.63 41.9 term. (B0015) gel extr. 2.48 0.01 1.9 * These results are aberrant. The absorption curve of the Nanodrop showed no peak around 280 nm. These non-nil results come from the absorption of other molecules in the media that have a peak around 230 nm. All of the DNA quantifications after gel extraction can be considered equal to 0, there's almost no DNA in these samples. Samples from 07/15 Sample name 260/280 260/230 ng/μL t/attC/mRFP restr. 0.60 0.29 19.8 t/attC/mRFP gel extr. -1.30 0.00 0.2 attC-t/attC/mRFP lig. 2.94 2.45 662.5* RBS/GFP/term (E0240) mini 1.89 1.74 174.5 RBS/GFP/term (E0240) restr. 0.75 0.34 20.9 RBS/GFP/term (E0240) gel extr. 1.19 0.01 0.9 RBS/GFP/term (E0240) lig. 2.94 2.60 664.3* * These results are aberrant. There's almost no DNA in these samples, the blank with only ligase buffer gives the same result : the T4 ligase buffer absorbs at 260 nm. In general, all these results explain why our transformations never worked. In fact, we don't have a lot of DNA from the beginning and we lose too much diring the experiments, especially gel extraction. So we don't have enough DNA for the ligation and so there's no colonies after transformation. The minipreps's yield is around 6μg DNA from a 10ml culture for a high copy number plasmid (pSB1A2) and around 3.5μg DNA from a 10ml culture for a lower copy number plasmid (pSU1ib). Using 10μL of the total 60μL miniprep for the digestion (total reaction volume of 50μL), we have about 1μg of DNA in our restriction digest reaction, so we don't lose anything. However, this is the total amount of DNA (both fragments). When loading the restriction digest into agarose gel for electrophoresis, we load only 17μL per well into 2 wells (we use only around 70% of our DNA). After electrophoresis and gel purification, we lose all the DNA. To have more DNA, we can use all the miniprep for digestion, load the maximum into 2 wells, purify both wells on the same column to concentrate all the DNA in 30μL and then use this DNA for ligation. Raphaël Restriction digest Using all 30 μL of DNA for the reaction to get more DNA digest. Incubation 1 hour at 37°C. attC with SpeI and Pst I (Buffer2) t/attC/mRFP with XbaI and PstI (B3) RBS/pLux (R0062) with SpeI and PstI (B2) Pm (J23110) with SpeI and PstI (B2) Ps (J23119) with SpeI and PstI (B2) RBS/LuxR (J37033) with XbaI and PstI (B3) RBS/LuxI (K081008) with EcoRI ans SpeI (B2) term (B0015) with EcoRI-HF and XbaI (B4) Gel electrophoresis Two agarose (0.8% w/v) gels using TAE buffer, no EtB in the gel, only EtB bath before UV revelation and cutting. 17μL restriction product + 3μL loading buffer/well ladder attC t/attC/mRFP pLux GFP Pmed Pstrong LuxR LuxI term Gel extraction with the new Qiagen kit Both gels are purified on the same column which allows us to concentrate all the DNA we obtained in only 30μL of water. Aleksandra Quantification of DNA with the Nanodrop Samples from 07/20 prepared from the 07/17 miniprep Sample name 260/280 260/230 ng/μL RBS/GFP/term (E0240) restr. 1.36 0.95 55.1 RBS/GFP/term (E0240) gel extr. 1.76 0.15 15.6 pLux (R0062) restr. 1.18 0.80 71.1 pLux (R0062) gel extr. 1.92 0.20 28.1 term/attC/mRFP restr. 1.23 0.76 48.2 term/attC/mRFP gel extr. 3.95 0.04 2.4* attC restr. 1.03 0.59 51.8 attC gel extr. 1.96 0.08 8.0* Pm (J23110) restr. 1.57 1.38 174.3 Pm (J23110) gel extr. 1.90 0.32 52.2 Ps (J23119) restr. 1.18 0.80 68.5 Ps (J23119) gel extr. 1.93 0.15 22.4 RBS/LuxI (K081008) restr. 1.20 0.57 76.1 RBS/LuxI (K081008)gel extr. 1.85 0.12 10.3 term (B0015) restr. 1.44 0.96 113.9 term (B0015) gel extr. 1.94 0.18 41.0 *These samples have too low DNA amount for the ligation, but we'll try anyway. In general these results show that the last miniprep (07/17) gave us almost DNA to work with, as we managed to have more DNA after the restriction digest and concentrate it during gel purification, even though we've lost a lot. The minipreps's yield is around 6μg DNA from a 10ml culture for a high copy number plasmid (pSB1A2) and around 3.5μg DNA from a 10ml culture for a lower copy number plasmid (pSU1ib). Using 30μL of the total 60μL miniprep for the digestion (total reaction volume of 50μL), we have about 3.5μg of DNA in our restriction digest reaction, so we don't lose anything. However, this is the total amount of DNA (both fragments). When loading the restriction digest into agarose gel for electrophoresis, we load only 17μL per well into 2 wells (we use only around 70% of our DNA). After electrophoresis and gel purification, the yield is about 0.7 to 1.5μg of a vector (we lose about 60% of DNA during these steps). The yield of an insert is lower because its DNA is smaller, so we have around around 0.3 to 0.5μg of an insert. This isn't enough DNA, but it might be enough for the ligation. Ligation overnight at 16°C We've tried to approach the perfect ligation conditions (about 500ng of the insert and the 1:3 vector:insert ratio. We've only had about 25μL of each DNA sample, so that's what we could do: LuxI(I) and term (V) : 25μL LuxI (250 ng) + 2μL term (82 ng) + 3.2 μL 10x ligation buffer + 1 μL T4 ligase attC (V) and t/attC/mRFP (I) : 25μL mRFP (60 ng) + 3μL attC (24 ng) + 3.2μL 10x ligation buffer + 1μL T4 ligase pLux (V) and GFP (I) : 25μL GFP (390 ng) + 4μL pLux (112ng) + 3.3μL 10xligation buffer + 1μL T4 ligase Raphaël Overnight cultures of the biobrick transformations from 07/19: J732006 (LacZ) - Amp B0031 (weak RBS) - Amp B0032 (medium RBS) - Amp B0034 (standard RBS) - Amp J31007 (TetA C) - Amp C0062 (LuxR w/o RBS) - Amp J37033 (RBS/LuxR) from the 2007 distribution plate - Amp Overnight cultures of the biobrick transformations from 07/19: J23119 (Ps) - Amp J23110 (Pm) - Amp attC -Kan term/attC/mRFP -Kan B0015 (term) - Amp E0240 (RBS/GFP/term) - Amp K081008 (LuxI) - Amp R0062 (pLux) - Amp Miniprep Of the overnight culture from one of the colonies that grew on one of the Kan transformation plates (attc-term/attC/mRFP) of 07/15. The liquid culture grew well in Kanamycin, so it wasn't a contaminant non-Kan resiatent colony that appeared on the plate after the kanamycin broke down with time. We want to check if these colonies are our successful transformations.