|
|
| Line 1: |
Line 1: |
| - | {{CSS/Delft2014_main}}
| |
| - | {{CSS/960_12_col}}
| |
| - | {{:Team:TU_Delft-Leiden/Templates/Start}}
| |
| | | | |
| - | <html>
| |
| - | <h2> Landmine Labjournal </h2>
| |
| - | <p> This labjournal will give more information on the cloning and characterisation
| |
| - | experiments performed within the landmine detection module.<br>
| |
| - | To go back to the content page click on the following link: </p>
| |
| - | <a href="https://2014.igem.org/Team:TU_Delft-Leiden/Project/Notebook">
| |
| - | <p> Content page notebook </p> </a>
| |
| - | <h3> 1st of July </h3>
| |
| - | <p> Grow on shakeflasks the strains received from Belkin lab (J::GFP, F::GFP, J::lux, F::lux, FB2::lux, FB2A1::lux)
| |
| - | and the host E. coli strain C43(DE3). F is the promoter from the yqjF gene from Escherichia coli.
| |
| - | J is the promoter from the ybiJ gene from Escherichia coli.
| |
| - | FB2 and FB2A1 are improved versions of the yqjF promoter generated via mutagenesis. </p>
| |
| - | <h3> 2nd of July </h3>
| |
| - | <p> Miniprep Belkin samples (J::GFP, F::GFP, J::lux, F::lux, FB2::lux, FB2A1::lux) <br>
| |
| - | Glycerol stock of Belkin samples (same as above) and the host C43(DE3) </p>
| |
| - | <h3> 16th of July </h3>
| |
| - | <p> Miniprep + Glycerol Stocks of mKate2. <br>
| |
| - | Measured the concentration of the isolated DNA with nanodrop:<br>
| |
| - | • mKate2 39.2 ng/ul </p>
| |
| - | <h3> 22th of July </h3>
| |
| - | <table border="1" style="width:50%">
| |
| - | <tr>
| |
| - | <td><b> PCR ybiJ from Belkin ybiJ:lux </b></td>
| |
| - | <td><b> Amount </b></td>
| |
| - | <td><b> Use </b></td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td>Template: ybiJ:lux [233 ng/ul]</td>
| |
| - | <td>75 ng</td>
| |
| - | <td>0.35 ul</td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td>FW ybiJ (5 uM)</td>
| |
| - | <td>5 pM</td>
| |
| - | <td>2.5 ul</td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td>RV ybiJ (5 uM)</td>
| |
| - | <td>5 pM</td>
| |
| - | <td>2.5 ul</td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td>dNTP (10 mM)</td>
| |
| - | <td></td>
| |
| - | <td>1.5 ul</td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td>pFx</td>
| |
| - | <td></td>
| |
| - | <td>0.2 ul</td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td>Buffer</td>
| |
| - | <td>10x</td>
| |
| - | <td>5 ul</td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td>MgSO<sub>4</sub> (50 mM)</td>
| |
| - | <td></td>
| |
| - | <td>1 ul</td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td>Enhancer</td>
| |
| - | <td></td>
| |
| - | <td>5 ul</td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td>MilliQ</td>
| |
| - | <td></td>
| |
| - | <td>32 ul</td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td>Total volume</td>
| |
| - | <td></td>
| |
| - | <td>50 ul</td>
| |
| - | </tr>
| |
| - | </table>
| |
| - | <p> Miniprepped the samples cultivated on 15.07.2014, except for the strain AYCE189.
| |
| - | Measured the concentration of the isolated DNA with nanodrop: <br>
| |
| - | PAYC002 122.8 ng/ul <br>
| |
| - | rr12y(rii)g 52.6 ng/ul<br>
| |
| - | PAYC003 187.0 ng/ul<br>
| |
| - | rrjt12(11)g 32.3 ng/ul<br>
| |
| - | PAYC005 27.8 ng/ul<br>
| |
| - | PAYC008 22.3 ng/ul<br>
| |
| - | PAYC006 37.0 ng/ul<br>
| |
| - | PAYC007 22.3 ng/ul<br>
| |
| - | I5023 22.5 ng/ul<br>
| |
| - | C640 14.3 ng/ul<br>
| |
| - | mKate 39.2 ng/ul<br>
| |
| - | eGFP 165.1 ng/ul<br>
| |
| - | Rhamnose 47.7 ng/ul<br>
| |
| - | Cultivated the samples again in shakeflasks.
| |
| - | Made glycerolstocks of all the cultivated samples from 15.07.2014.<br> <br>
| |
| - | Test the competency of C43(DE3): <br>
| |
| - | 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>
| |
| - | 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>
| |
| - | <h3> 17th of July </h3>
| |
| - | <p> Results of the plates for positive and negative control of C43<br>
| |
| - | • C43 + pUC19(AmpR) + Amp plates Much growth<br>
| |
| - | • C43 + pUC19(AmpR) + Cm plates No growth<br>
| |
| - | • C43 + pUC19(AmpR) + without antibiotic Much growth<br>
| |
| - | • 2x diluted C43 + pUC19(AmpR) + Amp plates Single colonies<br>
| |
| - | • 2x diluted C43 + pUC19(AmpR) + Cm plates No growth<br>
| |
| - | • 2x diluted C43 + pUC19(AmpR) + without antibiotic Much growth<br>
| |
| - | • C43 + no plasmid + Amp plates No growth<br>
| |
| - | • C43 + no plasmid + Kan plates No growth<br>
| |
| - | • C43 + no plasmid + Cm plates No growth<br>
| |
| - |
| |
| - | We decided to not dilute the competent cells for the future experiments.<br>
| |
| - | CFU for pUC19(AmpR): 1120 colonies on the plate.<br>
| |
| - |
| |
| - | Test the competency of BL21(DE3)<br>
| |
| - | • 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>
| |
| - | • 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>
| |
| - | • 2x diluted 30 ul competent BL21 cells + 100 ng pUC19 (AmpR)(190 ng/ul) plated in duplo on Amp (pos. control) LB plates </p>
| |
| - | <h3> 18th of July </h3>
| |
| - | <p> Results of the plates for positive and negative control of BL21(DE3)<br>
| |
| - | • BL21 + pUC19(AmpR) + Amp plates Much growth<br>
| |
| - | • BL21 + pUC19(AmpR) + Cm plates No growth<br>
| |
| - | • BL21 + pUC19(AmpR) + without antibiotic Much growth<br>
| |
| - | • 2x diluted BL21 + pUC19(AmpR) + Amp plates Single colonies<br>
| |
| - | • BL21 + no plasmid + Amp plates No growth<br>
| |
| - | • BL21 + no plasmid + without antibiotic Much growth<br>
| |
| - | • BL21 + no plasmid + Cm plates No growth<br> </p>
| |
| - | <h3> 22th of July </h3>
| |
| - | <p> Prepared antibiotics, 500ul in each eppendorf cup. Saved in ‘Antibiotics’ box in the freezer with other iGEM materials. 1000x stock solutions.<br>
| |
| - | • Chloramphenicol diluted in EtOH <br>
| |
| - | 34 mg/ml in EtOH -> 0,391 gram diluted in 11,5 ml EtOH<br>
| |
| - | • Kanamycin diluted in H2O<br>
| |
| - | 10 mg/ml in H2O -> 0,095 gram diluted in 9,5 ml H2O<br>
| |
| - | • Ampicillin diluted in H2O<br>
| |
| - | 100 mg/ml in H2O -> 0,92 gram diluted in 9,2 ml H2O<br>
| |
| - |
| |
| - | 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>
| |
| - | <h3> 23th of July </h3>
| |
| - | <p> Made competent cells of DH5a and saved in the freezer (-80). </p>
| |
| - | <h3> 28th of July </h3>
| |
| - | <p> The samples asked from Exeter iGEM team were prepared and sent to them. The samples requested are:
| |
| - | - BBa_K1022115 , Kanamycin resistant
| |
| - | - BBa_K1022105 , Chloramphenicol resistant
| |
| - | - BBa_K112808 , Ampicillin resistant
| |
| - |
| |
| - | Transformation of pUC19 in BL21 and DH5a as a control
| |
| - | • 30ul + 100ng pUC19 (190 ng/ul)
| |
| - | o Plated 50ul and 100 ul on Cam, Amp and plates without antibiotics
| |
| - | • 30ul competent cells
| |
| - | o Plated 50ul and 100 ul on Cam, Amp and plates without antibiotics</p>
| |
| - | <h3> 29th of July </h3>
| |
| - | <p> Cristy and Anne
| |
| - | Results of the DH5a and BL21 plates:
| |
| - | • BL21 + pUC19(AmpR) + Amp plates Much growth, big single colonies
| |
| - | • BL21 + pUC19(AmpR) + Cm plates No growth
| |
| - | • BL21 + pUC19(AmpR) + without antibiotic Much growth
| |
| - | • BL21 + no plasmid + Amp plates No growth
| |
| - | • BL21 + no plasmid + without antibiotic Much growth
| |
| - | • BL21 + no plasmid + Cm plates No growth
| |
| - | • DH5a + pUC19(AmpR) + Amp plates Much growth, little single colonies
| |
| - | • DH5a + pUC19(AmpR) + Cm plates No growth
| |
| - | • DH5a + pUC19(AmpR) + without antibiotic Much growth
| |
| - | • DH5a + no plasmid + Amp plates No growth
| |
| - | • DH5a + no plasmid + without antibiotic Much growth
| |
| - | • DH5a + no plasmid + Cm plates No growth
| |
| - | Tomek and Esra: making trace elements solution 2 attempts</p>
| |
| - | <h3> 30th of July </h3>
| |
| - | <p> Esra
| |
| - | Making the M4 minimal medium Trace Elements Solution (third attempt) + buffer (exact contents will be updated)
| |
| - | -Changed adding order
| |
| - | -Adding order 1- EDTA, 2- Mg, 3-Mn, 4-NaCl, 6-CoCl etc.
| |
| - | -Added number 5- (FeCl diluted in 22.5 mL HCl) at the end!!
| |
| - | -All added except FeCl (in HCl) -> no precipitations!! All is diluted well!
| |
| - |
| |
| - | Inoculated the transformed iGEM registry constructs
| |
| - | Resistance Transformation Grown in (ml LB):
| |
| - | Cam DH5a + k823017 5ml & 10 ml
| |
| - | Cam DH5a + k808000 5ml & 10 ml
| |
| - | Kan DH5a + I20260 5ml & 10 ml
| |
| - | Cam DH5a + 80017 5ml & 10 ml
| |
| - | Amp DH5a + J231100 5ml & 10 ml
| |
| - | Amp DH5a + pUC19 5ml & 10 ml</p>
| |
| - | <h3> 20th of August </h3>
| |
| - | <p> Janna
| |
| - | Made medium M4 with different carbon sources.
| |
| - | 1. 400 ml 40 mM D/L-lactate M4
| |
| - | 342 ml MilliQ
| |
| - | 10 ml Buffer 40 x
| |
| - | 4 ml 0.1 M CaCl2
| |
| - | 40 ml 0.4 M D/L-lactate
| |
| - | 4 ml Trace elements 100 x
| |
| - | 2. 400 ml 40 mM glycerol M4
| |
| - | 342 ml MilliQ
| |
| - | 10 ml Buffer 40 x
| |
| - | 4 ml 0.1 M CaCl2
| |
| - | 40 ml 0.4 M Glycerol
| |
| - | 4 ml Trace elements 100 x
| |
| - | 3. 400 ml 40 mM glucose M4
| |
| - | 342 ml MilliQ
| |
| - | 10 ml Buffer 40 x
| |
| - | 4 ml 0.1 M CaCl2
| |
| - | 40 ml 0.4 M Glucose
| |
| - | 4 ml Trace elements 100 x </p>
| |
| - | <h3> 21th of August </h3>
| |
| - | <p> Janna
| |
| - | Tested competent cells BL21 culture 1, BL21 culture 2 and C43. I used pUC19 as test DNA (ampR). Made the following combinations:
| |
| - | 1. 30 μl BL21.1 with 1 μl MilliQ
| |
| - | 2. 30 μl BL21.1 with 1 μl pUC19
| |
| - | 3. 30 μl BL21.2 with 1 μl MilliQ
| |
| - | 4. 30 μl BL21.2 with 1 μl pUC19
| |
| - | 5. 30 μl C43 with 1 μl MilliQ
| |
| - | 6. 30 μl C43 with 1 μl pUC19
| |
| - |
| |
| - | There were no colonies on the plates, so the cells are not competent.
| |
| - | Cristy: Made competent cells of CsgB (approximately 30 eppendorfs containing 100ul competent cells). OD600: 0,567 and 0,589</p>
| |
| - | <h3> 22th of August </h3>
| |
| - | <p> Joan
| |
| - | Prepare samples for Melbourne iGEM team:
| |
| - | • pET23b - Ulp1-His6 (AmpR)
| |
| - | • BBa_K1022107:pcI-Ulp in pSB1C3 col2
| |
| - | • BBa_K1022113:pBAD-Ulp-TT in pSB1C3 col2 </p>
| |
| - | <h3> 26th of August </h3>
| |
| - | <p> Chloramphenicol diluted in EtOH
| |
| - | 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.
| |
| - |
| |
| - | Janna
| |
| - | Making M4 with D/L-lactate. Same protocol as 20/8, only filter sterilized the whole bottle after making it.</p>
| |
| - | <h3> 28th of August </h3>
| |
| - | <p> Janna
| |
| - | Transformation of pUC19 in C43+ET20 as test
| |
| - | Made the following transformations:
| |
| - | 0.5 ul pUC19 plasmid (concentration of 100.2 ng/ul) added to 30 ul of C43+ET20 competent cells
| |
| - | 1 ul MilliQ added to 30 ul of C43+ET20 competent cells as a negative control
| |
| - |
| |
| - | Followed the transformation in home-made competent cells protocol and made six plates with each 100 ul:
| |
| - | pUC19 in C43+ET20 (ampR and camR):
| |
| - | Ampicillin - some growth
| |
| - | Chloramphenicol - some growth
| |
| - | Amp and Cam - lots of growth
| |
| - | MQ in C43+ET20 (camR):
| |
| - | Ampicillin - no growth
| |
| - | Chloramphenicol - some growth
| |
| - | Amp and Cam - no growth
| |
| - |
| |
| - | This means the cells are competent, but the efficiency is low.</p>
| |
| - |
| |
| - | </html>
| |
| - |
| |
| - | {{:Team:TU_Delft-Leiden/Templates/End}}
| |
"
