Team:TU Delft-Leiden/Project/Notebook

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    <h2> General Labjournal </h2>
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<p> This labjournal will give more information on the production of stocks, general testing not within a certain module and
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collaborations with other iGEM teams.<br>
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To go back to the content page click on the following link: </p>
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<a href="https://2014.igem.org/Team:TU_Delft-Leiden/Project/Notebook">
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        <p> Content page notebook </p> </a>
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<h3> 25th of June </h3>
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<p> Grow on shakeflasks the 25 samples that we requested from the iGEM HQ.
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The biobrick numbers of the requested samples: K1172305, K1172306, K1172401, K1172403,
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K1172404, K917013, K917014, K1127006, K1172303, K1172304, K1179019, K917012, K917009,
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K917006, K917003, K1172405, K1172501, K1172502, K1172503, K1172504, K1172505, K1172507,
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K540000, K342003, K540001. </p>
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<h3> 26th of June </h3>
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<p> Miniprepped and made glycerol stocks of the 25 samples that we requested from the iGEM HQ. </p>
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<h3> 7th of July </h3>
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<p> Prepare solutions for competent cells preparation (MgCl2 and CaCl2) </p>
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<h3> 8th of July </h3>
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<p> Prepare all the sequencing mixtures. The parts to be sequenced were:
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K1172306, K1172403, K1172404, K1172303, K917009, K917006, K917003, K1172502, K540000 and K342003. </p>
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    <h3> 15th of July </h3>
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<p> Made competent cells of C43(DE3) and BL21(DE3) for transformation.
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Cultivate the transformed constructs from 14.07.2014 in LB medium for miniprep.
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Also cultivate mKate (amp), eGFP (amp) and Rhamnose promoter - Bba_K914003 (cam)
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and the strain AYCE189 from Lu lab is also cultivated (for a glycerolstock). </p>
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<h3> 16th of July </h3>
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<p> Miniprepped the samples cultivated on 15.07.2014, except for the strain AYCE189.
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Measured the concentration of the isolated DNA with nanodrop: <br>
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PAYC002 122.8 ng/ul <br>
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rr12y(rii)g 52.6 ng/ul<br>
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PAYC003 187.0 ng/ul<br>
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rrjt12(11)g 32.3 ng/ul<br>
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PAYC005 27.8 ng/ul<br>
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PAYC008 22.3 ng/ul<br>
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PAYC006 37.0 ng/ul<br>
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PAYC007 22.3 ng/ul<br>
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I5023 22.5 ng/ul<br>
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C640 14.3 ng/ul<br>
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mKate 39.2 ng/ul<br>
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eGFP 165.1 ng/ul<br>
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Rhamnose 47.7 ng/ul<br>
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Cultivated the samples again in shakeflasks.
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Made glycerolstocks of all the cultivated samples from 15.07.2014.<br> <br>
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Test the competency of C43(DE3): <br>
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30 ul competent C43 cells + 100 ng pUC19 (AmpR)(190 ng/ul), plated in duplo on Cm (neg. control), Amp (pos. control) LB plates and on LB plate without antibiotics<br>
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30 ul competent C43 cells plated in duplo on Cm (neg. control), Amp (neg. control) LB plates and on LB plate without antibiotics (pos. control)</p>
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<h3> 17th of July </h3>
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<p> Results of the plates for positive and negative control of C43<br>
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• C43 + pUC19(AmpR) + Amp plates Much growth<br>
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• C43 + pUC19(AmpR) + Cm plates No growth<br>
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• C43 + pUC19(AmpR) + without antibiotic Much growth<br>
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• 2x diluted C43 + pUC19(AmpR) + Amp plates Single colonies<br>
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• 2x diluted C43 + pUC19(AmpR) + Cm plates No growth<br>
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• 2x diluted C43 + pUC19(AmpR) + without antibiotic Much growth<br>
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• C43 + no plasmid + Amp plates No growth<br>
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• C43 + no plasmid + Kan plates No growth<br>
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• C43 + no plasmid + Cm plates No growth<br>
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We decided to not dilute the competent cells for the future experiments.<br>
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<html>
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CFU for pUC19(AmpR): 1120 colonies on the plate.<br>
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<body>
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<div class='grid_12'>
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Test the competency of BL21(DE3)<br>
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<h2> Notebook </h2>
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• 30 ul competent BL21 cells + 100 ng pUC19 (AmpR)(190 ng/ul), plated in duplo on Cm (neg. control), Amp (pos. control) LB plates and on LB plate without antibiotics<br>
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• 30 ul competent BL21 cells plated in duplo on Cm (neg. control), Amp (neg. control) LB plates and on LB plate without antibiotics (pos. control)<br>
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• 2x diluted 30 ul competent BL21 cells + 100 ng pUC19 (AmpR)(190 ng/ul) plated in duplo on Amp (pos. control) LB plates </p>
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<h3> 18th of July </h3>
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<p> Results of the plates for positive and negative control of BL21(DE3)<br>
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• BL21 + pUC19(AmpR) + Amp plates Much growth<br>
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• BL21 + pUC19(AmpR) + Cm plates No growth<br>
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• BL21 + pUC19(AmpR) + without antibiotic Much growth<br>
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• 2x diluted BL21 + pUC19(AmpR) + Amp plates Single colonies<br>
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• BL21 + no plasmid + Amp plates No growth<br>
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• BL21 + no plasmid + without antibiotic Much growth<br>
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• BL21 + no plasmid + Cm plates No growth<br> </p>
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<h3> 22th of July </h3>
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<p> Prepared antibiotics, 500ul in each eppendorf cup. Saved in ‘Antibiotics’ box in the freezer with other iGEM materials. 1000x stock solutions.<br>
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• Chloramphenicol diluted in EtOH <br>
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34 mg/ml in EtOH -> 0,391 gram diluted in 11,5 ml EtOH<br>
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• Kanamycin diluted in H2O<br>
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10 mg/ml in H2O -> 0,095 gram diluted in 9,5 ml H2O<br>
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• Ampicillin diluted in H2O<br>
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100 mg/ml in H2O -> 0,92 gram diluted in 9,2 ml H2O<br>
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Pre-culture DH5-alpha has been prepared, 100 mL LB medium is cultivated and placed in the shaker. Overnight at 37 degrees and 180 rpm. </p>
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<p>
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<h3> 23th of July </h3>
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In this notebook you can find all details on protocols, cloning and characterization experiments.
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<p> Made competent cells of DH5a and saved in the freezer (-80). </p>
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Every link is a download link for a pdf file, be aware that the pdf files of the labjournals are quite large.  
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<h3> 28th of July </h3>
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</p>
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<p> The samples asked from Exeter iGEM team were prepared and sent to them. The samples requested are:
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- BBa_K1022115 , Kanamycin resistant
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- BBa_K1022105 , Chloramphenicol resistant
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- BBa_K112808 , Ampicillin resistant
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Transformation of pUC19 in BL21 and DH5a as a control
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<br>
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• 30ul + 100ng pUC19 (190 ng/ul)
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o Plated 50ul and 100 ul on Cam, Amp and plates without antibiotics
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• 30ul competent cells
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o Plated 50ul and 100 ul on Cam, Amp and plates without antibiotics</p>
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<h3> 29th of July </h3>
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<p> Cristy and Anne
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Results of the DH5a and BL21 plates:
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• BL21 + pUC19(AmpR) + Amp plates Much growth, big single colonies
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• BL21 + pUC19(AmpR) + Cm plates No growth
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• BL21 + pUC19(AmpR) + without antibiotic Much growth
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• BL21 + no plasmid + Amp plates No growth
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• BL21 + no plasmid + without antibiotic Much growth
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• BL21 + no plasmid + Cm plates No growth
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• DH5a + pUC19(AmpR) + Amp plates Much growth, little single colonies
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• DH5a + pUC19(AmpR) + Cm plates No growth
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• DH5a + pUC19(AmpR) + without antibiotic Much growth
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• DH5a + no plasmid + Amp plates No growth
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• DH5a + no plasmid + without antibiotic Much growth
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• DH5a + no plasmid + Cm plates No growth
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Tomek and Esra: making trace elements solution 2 attempts</p>
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<h3> 30th of July </h3>
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<p> Esra
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Making the M4 minimal medium Trace Elements Solution (third attempt) + buffer (exact contents will be updated)
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-Changed adding order
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-Adding order 1- EDTA, 2- Mg, 3-Mn, 4-NaCl, 6-CoCl etc.
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-Added number 5- (FeCl diluted in 22.5 mL HCl) at the end!!
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-All added except FeCl (in HCl) -> no precipitations!! All is diluted well!
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Inoculated the transformed iGEM registry constructs
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<h3>Labjournals</h3>  
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Resistance Transformation Grown in (ml LB):
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Cam DH5a + k823017 5ml & 10 ml
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Cam DH5a + k808000 5ml & 10 ml
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Kan DH5a + I20260 5ml & 10 ml
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Cam DH5a + 80017 5ml & 10 ml
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Amp DH5a + J231100 5ml & 10 ml
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Amp DH5a + pUC19 5ml & 10 ml</p>
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<h3> 20th of August </h3>
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<p> Janna
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Made medium M4 with different carbon sources.
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1. 400 ml 40 mM D/L-lactate M4
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342 ml MilliQ
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10 ml Buffer 40 x
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4 ml 0.1 M CaCl2
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40 ml 0.4 M D/L-lactate
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4 ml Trace elements 100 x
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2. 400 ml 40 mM glycerol M4
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342 ml MilliQ
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10 ml Buffer 40 x
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4 ml 0.1 M CaCl2
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40 ml 0.4 M Glycerol
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4 ml Trace elements 100 x
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3. 400 ml 40 mM glucose M4
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342 ml MilliQ
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10 ml Buffer 40 x
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4 ml 0.1 M CaCl2
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40 ml 0.4 M Glucose
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4 ml Trace elements 100 x </p>
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<h3> 21th of August </h3>
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<p> Janna
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Tested competent cells BL21 culture 1, BL21 culture 2 and C43. I used pUC19 as test DNA (ampR). Made the following combinations:
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1. 30 μl BL21.1 with 1 μl MilliQ
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2. 30 μl BL21.1 with 1 μl pUC19
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3. 30 μl BL21.2 with 1 μl MilliQ
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4. 30 μl BL21.2 with 1 μl pUC19
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5. 30 μl C43 with 1 μl MilliQ
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6. 30 μl C43 with 1 μl pUC19
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There were no colonies on the plates, so the cells are not competent.
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<p><a href="https://static.igem.org/mediawiki/2014/2/29/Delft2014_Labjournal_General.pdf">
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Cristy: Made competent cells of CsgB (approximately 30 eppendorfs containing 100ul competent cells). OD600: 0,567 and 0,589</p>
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                    <p>General work </p>
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<h3> 22th of August </h3>
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                    </a></p>
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<p> Joan
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<p><a href="https://static.igem.org/mediawiki/2014/1/10/Delft2014_LabjournalLandmine.pdf">
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Prepare samples for Melbourne iGEM team:
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                    <p>Landmine Module</p>
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• pET23b - Ulp1-His6 (AmpR)
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                    </a></p>
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• BBa_K1022107:pcI-Ulp in pSB1C3 col2
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<p><a href="https://static.igem.org/mediawiki/2014/b/b8/Delft2014_LabjournalEET.pdf">
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• BBa_K1022113:pBAD-Ulp-TT in pSB1C3 col2 </p>
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                    <p>EET Module</p>
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<h3> 26th of August </h3>
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                    </a></p>
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<p> Chloramphenicol diluted in EtOH
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<p> <a href="https://static.igem.org/mediawiki/2014/a/ae/Delft2014_LabjournalCurli.pdf">
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34 mg/ml in EtOH -> 0,3912 gram diluted in 11,5ml EtOH. Divided in aliquots of 500 ul. Refilled stock with 22 new cups.
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                    <p>Curli Module</p>
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                    </a></p>
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<br>
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Janna
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<h3>Protocols characterization</h3>
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Making M4 with D/L-lactate. Same protocol as 20/8, only filter sterilized the whole bottle after making it.</p>
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<p><a href="https://static.igem.org/mediawiki/2014/d/d9/Delft2014_Hemestainingprotocol.pdf">
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<h3> 28th of August </h3>
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                    <p>Heme stain protocol</p>
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<p> Janna
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                    </a></p>
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Transformation of pUC19 in C43+ET20 as test
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<p><a href="https://static.igem.org/mediawiki/2014/9/92/Delft2014_Membraneproteinpurification.pdf">
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Made the following transformations:
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                    <p>Membrane protein purification</p>
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0.5 ul pUC19 plasmid (concentration of 100.2 ng/ul) added to 30 ul of C43+ET20 competent cells
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                    </a></p>
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1 ul MilliQ added to 30 ul of C43+ET20 competent cells as a negative control
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<p><a href="https://static.igem.org/mediawiki/2014/a/a1/Delft2014_SDS-PAGEprotocol.pdf">
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                    <p>SDS-PAGE</p>
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                    </a></p>
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<p><a href="https://static.igem.org/mediawiki/2014/f/f4/Delft2014_PlatereaderLD.pdf">
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                    <p>Plate reader Landmine Detection</p>
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                    </a></p>
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<p><a href="https://static.igem.org/mediawiki/2014/1/16/Delft2014_curliplatereader.pdf">
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                    <p>Plate reader Conductive Curli</p>
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                    </a></p>
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<p><a href="https://static.igem.org/mediawiki/2014/7/7a/Delft2014_ProtocolCongoRed.pdf">
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                    <p>Congo Red protocol</p>
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                    </a></p>
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<p><a href="https://static.igem.org/mediawiki/2014/8/83/Delft2014_Gold_NP_and_CV_assay.pdf">
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                    <p>Gold nanoparticles and crystal violet protocol</p>
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                    </a></p>
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<p><a href="https://static.igem.org/mediawiki/2014/d/dc/Delft2014_Bioreactor_protocol.pdf">
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                    <p>Bioreactor protocol</p>
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                    </a></p>
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<p><a href="https://static.igem.org/mediawiki/2014/8/83/TUDelft_2014_Protocol_Promoter_Characterisation_iGEM_Wageningen.pdf">
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                    <p>Wageningen 2014 iGEM team collaboration protocol</p>
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                    </a></p>
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Followed the transformation in home-made competent cells protocol and made six plates with each 100 ul:
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<h3> Protocols cloning </h3>
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pUC19 in C43+ET20 (ampR and camR):
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<p>    <a href="https://static.igem.org/mediawiki/2014/0/06/Delft2014_Miniprepprotocol.pdf">
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Ampicillin - some growth
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                    <p>Miniprep </p>
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Chloramphenicol - some growth
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                    </a></p>
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Amp and Cam - lots of growth
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<p><a href="https://static.igem.org/mediawiki/2014/a/a5/Delft2014_Electroporationprotocol.pdf">
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MQ in C43+ET20 (camR):
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                    <p>Electroporation</p>
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Ampicillin - no growth
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                    </a></p>
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Chloramphenicol - some growth
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<p><a href="https://static.igem.org/mediawiki/2014/e/e7/Delft2014_Gelelectrophoresis.pdf">
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Amp and Cam - no growth
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                    <p>Gel electrophoresis</p>
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                    </a></p>
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<p><a href="https://static.igem.org/mediawiki/2014/6/6c/Delft2014_Glycerolstockpreparation.pdf">
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                    <p>Glycerol stock preparation</p>
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                    </a></p>
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<p><a href="https://static.igem.org/mediawiki/2014/e/ed/Delft2014_Makingcompetentcells.pdf">
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                    <p>Home-made competent cells</p>
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                    </a></p>
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<p><a href="https://static.igem.org/mediawiki/2014/9/9b/Delft2014_MinElutegelextraction.pdf">
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                    <p>Extraction of DNA from gel</p>
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                    </a></p>
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<p><a href="https://static.igem.org/mediawiki/2014/9/96/Delft2014_PCRpurificationQIAquickkit.pdf">
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                    <p>PCR purification</p>
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                    </a></p>
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<p><a href="https://static.igem.org/mediawiki/2014/8/87/Delft2014_RestrictionofplasmidDNA.pdf">
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                    <p>Restriction of plasmid DNA</p>
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                    </a></p>
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<p><a href="https://static.igem.org/mediawiki/2014/0/00/Delft2014_Restriction_of_PCR_product.pdf">
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                    <p>Restriction of PCR product</p>
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                    </a></p>
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<p><a href="https://static.igem.org/mediawiki/2014/2/22/Delft2014_TransformationDH5alpha.pdf">
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                    <p>Transformation in commercial DH5alpha</p>
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                    </a></p>
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<p><a href="https://static.igem.org/mediawiki/2014/4/45/Delft2014_Transformationhomemade.pdf">
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                    <p>Transformation in home-made competent cells</p>
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                    </a></p>
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<p><a href="https://static.igem.org/mediawiki/2014/7/79/Delft2014_Trace_element_solution.pdf">
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                    <p>Trace element solution</p>
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                    </a></p>
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<p><a href="https://static.igem.org/mediawiki/2014/b/b1/Delft2014_Antibiotics.pdf">
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                    <p>Antibiotics stocks</p>
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                    </a></p>
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This means the cells are competent, but the efficiency is low.</p>
 
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</div>
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</body>
</html>
</html>
{{:Team:TU_Delft-Leiden/Templates/End}}
{{:Team:TU_Delft-Leiden/Templates/End}}

Latest revision as of 20:55, 17 October 2014


Notebook

In this notebook you can find all details on protocols, cloning and characterization experiments. Every link is a download link for a pdf file, be aware that the pdf files of the labjournals are quite large.


Labjournals

General work

Landmine Module

EET Module

Curli Module


Protocols characterization

Heme stain protocol

Membrane protein purification

SDS-PAGE

Plate reader Landmine Detection

Plate reader Conductive Curli

Congo Red protocol

Gold nanoparticles and crystal violet protocol

Bioreactor protocol

Wageningen 2014 iGEM team collaboration protocol

Protocols cloning

Miniprep

Electroporation

Gel electrophoresis

Glycerol stock preparation

Home-made competent cells

Extraction of DNA from gel

PCR purification

Restriction of plasmid DNA

Restriction of PCR product

Transformation in commercial DH5alpha

Transformation in home-made competent cells

Trace element solution

Antibiotics stocks

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