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| | <table width="100%" border="0"> | | <table width="100%" border="0"> |
| | <tr> | | <tr> |
| - | <td colspan="2"> {{UNIPV-Pavia/header}} </td> | + | <td colspan="3"> {{UNIPV-Pavia/header}} </td> |
| - | <!-- <td colspan=" 2" align="center"><html><img src="http://img337.imageshack.us/img337/7634/logoigem.jpg" width="100%" /></html></td> --> | + | <!-- <td colspan=" 3" align="center"><html><img src="http://img337.imageshack.us/img337/7634/logoigem.jpg" width="100%" /></html></td> --> |
| | </tr> | | </tr> |
| | | | |
| | {{UNIPV-Pavia/Style}} | | {{UNIPV-Pavia/Style}} |
| | | | |
| - | <tr><td align="left" valign="top" width="20%">{{UNIPV-Pavia/menu}}</td> | + | <tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td> |
| | <td valign="top"> | | <td valign="top"> |
| - | <table border="0" align="center" width="100%"><tr><td align="justify" valign="top"> | + | <table border="0" align="center" width="100%" class="trasparenza"><tr><td align="justify" valign="top" style="padding:20px"> |
| - | <html><p align="center"><font size="4"><b>JUNE</b></font></p></html><hr><br>
| + | |
| - | <table class=cont border="0" width="30%" align="center" valign="top">
| + | |
| - | <tr><th align="center" width="12%"><b>DATE</b></th><th align="center"><b>ACTIVITY</b></th></tr>
| + | |
| - | <tr><td align="center" valign="top">June, 3th</td><td align="left">9° Meeting - Discussion of final and technical details about [[Team:UNIPV-Pavia/Project|The Project]]. Planning of Bio-Lab activity.</td></tr> | + | |
| - | <tr><td align="center" valign="top">[[#June, 7th|June, 7th]]</td><td align="left"><partinfo>BBa_E2050</partinfo>, <partinfo>BBa_K165018</partinfo>, <partinfo>BBa_K165037</partinfo>, <partinfo>BBa_J61001</partinfo>, <partinfo>BBa_K081008</partinfo> and <partinfo>BBa_K125500</partinfo> were resuspended from Spring 2010 DNA distribution. <partinfo>BBa_P1004</partinfo> was resuspended from Spring 2009 DNA distribution. All BioBricks were transformed in ''E. coli'' DH5alpha.<br>Liquid LB+Amp and LB+Amp agar plates were prepared according with "protocols".</td></tr>
| + | |
| - | <tr><td align="center" valign="top">[[#June, 8th|June, 8th]]</td><td align="left">Inoculum from plates in liquid Lb and glycerol stocks preparation. Falcon were re-filled for tomorrow mini-prep<br></td></tr>
| + | |
| - | <tr><td align="center" valign="top">[[#June, 9th|June, 9th]]</td><td align="left">MiniPrep of <partinfo>BBa_E2050</partinfo>, <partinfo>BBa_K165018</partinfo>, <partinfo>BBa_K165037</partinfo>, <partinfo>BBa_J61001</partinfo>, <partinfo>BBa_K081008</partinfo> and <partinfo>BBa_K125500</partinfo>. Digestion, gel run/cut and ligation of:
| + | |
| - | *I0: <partinfo>BBa_K125500</partinfo> (E-S)+<partinfo>BBa_B0015</partinfo> (E-X)
| + | |
| - | *I1: <partinfo>BBa_E2050</partinfo> (E-S)+<partinfo>BBa_K165018</partinfo> (E-X)
| + | |
| - | *I2: <partinfo>BBa_P1004</partinfo> (E-S)+<partinfo>BBa_B0015</partinfo> (E-X)
| + | |
| - | *I3: <partinfo>BBa_K081008</partinfo> (E-S)+<partinfo>BBa_B0015</partinfo> (E-X)<br></td></tr>
| + | |
| - | <tr><td align="center" valign="top">[[#June, 10th|June, 10th]]</td><td align="left">Transformation of ligations I0, I1, I2, I3 in home made ''E. coli'' DH5-alpha competent cells. Transformants were plated on LB+Amp agar plates<br></td></tr>
| + | |
| - | <tr><td align="center" valign="top">[[#June, 11th|June, 11th]]</td><td align="left">Inoculum from plates in liquid LB and glycerol stocks preparation for I0, I1, I2 and I3 in duplicate (2 colonies peaked from each plate).<br></td></tr>
| + | |
| - | <tr><td align="center" valign="top">June, 12th</td><td align="left">iGEM EU workshop<br></td></tr>
| + | |
| - | <tr><td align="center" valign="top">June, 13th</td><td align="left">iGEM EU workshop<br></td></tr>
| + | |
| - | <tr><td align="center" valign="top">[[#June, 14th|June, 14th]]</td><td align="left">6° Bio-Lab - Lorenzo and Nicolò.<br></td></tr>
| + | |
| - | <tr><td align="center" valign="top">[[#June, 15th|June, 15th]]</td><td align="left">7° Bio-Lab - Lorenzo and Manuel L., Sara and Alessandro.<br></td></tr>
| + | |
| - | <tr><td align="center" valign="top">[[#June, 16th|June, 16th]]</td><td align="left">8° Bio-Lab - Susanna Z., Sara and Nicolò.<br></td></tr>
| + | |
| - | <tr><td align="center" valign="top">[[#June, 17th|June, 17th]]</td><td align="left">9° Bio-Lab - Susanna Z., Nicolò and Riccardo.<br></td></tr>
| + | |
| - | <tr><td align="center" valign="top">[[#June, 18th|June, 18th]]</td><td align="left">10° Bio-Lab - Susanna Z., Susanna S. and Federica.<br></td></tr>
| + | |
| - | | + | |
| - | </table>
| + | |
| - | ==June, 7th==
| + | |
| - | These BioBrick were resuspended in 15ul ddH20:
| + | |
| - | *<partinfo>BBa_E2050</partinfo> (mOrange, 744bp) 2010 Kit Plate 2, well 13N in pSB2K3
| + | |
| - | *<partinfo>BBa_K165018</partinfo> (ADH1 terminator, 253 bp) 2010 Kit Plate 3, well 2M, J63009 (Amp)
| + | |
| - | *<partinfo>BBa_K165037</partinfo> (tef2 promoter, 403bp) 2010 Kit Plate 3, well 22O, pSB1AK3
| + | |
| - | *<partinfo>BBa_P1004</partinfo> (Chloramphenicol resistence cassette, 769 bp) 2009 Kit plate 1, well 7B, pSB1A1
| + | |
| - | *<partinfo>BBa_J61001</partinfo> (R6K origin, 406bp) 2010 Kit plate 1, well 240, pSB1A2
| + | |
| - | *<partinfo>BBa_K081008</partinfo> (RBS-luxI, 664bp) 2010 Kit plate 2, well 10L, pSB1A2
| + | |
| - | *<partinfo>BBa_K125500</partinfo> (GFP fusion brick, 718bp) 2010 Kit plate 3, well 2P, pSB1A2
| + | |
| - | | + | |
| - | 1,5 ul of each culture was transformed in 100 ul of home-made competent ''E. coli'' DH5alpha.
| + | |
| - | | + | |
| - | Tranformants were all plated on LB agar plates added with Ampicillin, except for <partinfo>BBa_E2050</partinfo> (Kanamycin).
| + | |
| - | | + | |
| - | 500 ml LB+Amp was prepared and 21 LB agar plates + Amp were prepared (500 ml).
| + | |
| - | | + | |
| - | ==June, 8th==
| + | |
| - | | + | |
| - | All plates grown overnight at 37°C show colonies!
| + | |
| - | In particular, <partinfo>BBa_E2050</partinfo> (grown on LB+Kan), <partinfo>BBa_K125500</partinfo> (grown on LB+Amp), <partinfo>BBa_K081008</partinfo> (grown on LB+Amp) and <partinfo>BBa_K165018</partinfo> (grown on LB+Amp) showed big colonies, well separated on the plate.
| + | |
| - | <partinfo>BBa_P1004</partinfo> (grown on LB+Amp) and <partinfo>BBa_J61001</partinfo> (grown on LB+Amp) showed many colonies, with big colonies surrounded by small colonies.
| + | |
| - | <partinfo>BBa_K165037</partinfo> (grown on LB+Amp) showed only 11 big colonies.
| + | |
| - | | + | |
| - | A colony was peaked for each plate and inoculated in 1ml LB+antibiotic.
| + | |
| - | | + | |
| | | | |
| - | <center>
| + | <table class="menu" border="0" width="100%"> |
| - | <table width='100%'> | + | |
| | <tr> | | <tr> |
| - | <td> | + | <td align="center"> |
| - | {|
| + | [[Team:UNIPV-Pavia/Calendar/June/settimana2|Week 2]] |
| - | | <partinfo>BBa_E2050</partinfo> || 1ml LB+Kan
| + | |
| - | |-
| + | |
| - | | <partinfo>BBa_K165037</partinfo> || 1ml LB+Amp
| + | |
| - | |-
| + | |
| - | | <partinfo>BBa_P1004</partinfo> || 1ml LB+Amp
| + | |
| - | |-
| + | |
| - | | <partinfo>BBa_K125500</partinfo> || 1ml LB+Amp
| + | |
| - | |-
| + | |
| - | | <partinfo>BBa_K081008</partinfo> || 1ml LB+Amp
| + | |
| - | |-
| + | |
| - | | <partinfo>BBa_J61001</partinfo> || 1ml LB+Amp
| + | |
| - | |-
| + | |
| - | | <partinfo>BBa_K165018</partinfo> || 1ml LB+Amp
| + | |
| - | |} | + | |
| | </td> | | </td> |
| - | <td> | + | <td align="center"> |
| - | <table>
| + | [[Team:UNIPV-Pavia/Calendar/June/settimana3|Week 3]] |
| - | <tr>
| + | |
| - | <td>
| + | |
| | </td> | | </td> |
| - | </tr>
| + | <td align="center"> |
| - | <tr>
| + | [[Team:UNIPV-Pavia/Calendar/June/settimana4|Week 4]] |
| - | <td> | + | |
| - | [[Image:UNIPV_attivita_lab2.jpg|thumb|300px|left|Sara and Nicolò at work]] | + | |
| | </td> | | </td> |
| | </tr> | | </tr> |
| | </table> | | </table> |
| - | </td>
| |
| - | </tr>
| |
| - | </table>
| |
| - | </center>
| |
| | | | |
| | | | |
| - | <partinfo>BBa_K165037</partinfo> in pSB1AK3 plasmid was inocultaed both in 1ml LB+Amp and in 1ml LB+Kan for a phenotipic assay. | + | <html><p align="center"><font size="4"><b>JUNE</b></font></p></html><hr><br> |
| - | | + | Finally Bio-Lab activity began!<br><br><font size="4"><b><u> Select the day you are interested in to find out all the details!</u></b></font><br><br> |
| - | Cultures were grown for 8 hours at 37°C 220 rpm.
| + | <table class="cont" border="0" width="30%" align="center" valign="top"> |
| - | | + | <tr><th align="center" width="12%"><b>DATE</b></th><th align="center"><b>ACTIVITY</b></th></tr> |
| - | After this time, cultures were all grown and in saturation phase. <partinfo> BBa_K165037</partinfo> in LB+Kan was also grown, so that this phenotipic assay was positive. | + | <tr><td align="center" valign="top"><html><a name="June_3rd"></a></html>June, 3rd</td><td align="left">9° Meeting - Discussion of final and technical details about [[Team:UNIPV-Pavia/Project|The Project]]. Planning of Bio-Lab activity.</td></tr> |
| - | | + | <tr><th align="center" width="12%" colspan="2"><b>JUNE: WEEK 2</b></th></tr> |
| - | Glycerol stocks were prepared for each culture (250ul saturated culture + 750 ul glycerol 80%) and stored at -80°C.
| + | <tr><td align="center" valign="top">[[Team:UNIPV-Pavia/Calendar/June/settimana2 #June, 7th|June, 7th]]</td><td align="left">1° Bio-Lab - <partinfo>BBa_E2050</partinfo>, <partinfo>BBa_K165018</partinfo>, <partinfo>BBa_K165037</partinfo>, <partinfo>BBa_J61001</partinfo>, <partinfo>BBa_K081008</partinfo> and <partinfo>BBa_K125500</partinfo> were resuspended from Spring 2010 DNA distribution. <partinfo> BBa_P1004</partinfo> was resuspended from Spring 2009 DNA distribution. All BioBricks were transformed in ''E. coli'' DH5alpha.<br>Liquid LB+ Amp and LB+Amp agar plates were prepared according with "protocols". </td></tr> |
| - | | + | <tr><td align="center" valign="top">[[Team:UNIPV-Pavia/Calendar/June/settimana2 #June, 8th|June, 8th]]</td><td align="left">2° Bio-Lab - Inoculum from plates in liquid Lb and glycerol stocks preparation. Falcon were re-filled for tomorrow mini-prep.<br></td></tr> |
| - | The left culture was re-filled to 5ml with LB+antibiotic and grown overnight at 37°C 200 rpm to be miniprepped tomorrow.
| + | <tr><td align="center" valign="top">[[Team:UNIPV-Pavia/Calendar/June/settimana2 #June, 9th|June, 9th]]</td><td align="left">3° Bio-Lab - MiniPrep of <partinfo>BBa_E2050</partinfo>, <partinfo>BBa_K165018</partinfo>, <partinfo>BBa_K165037</partinfo>, <partinfo>BBa_J61001</partinfo>, <partinfo>BBa_K081008</partinfo> and <partinfo>BBa_K125500</partinfo>. Digestion, gel run/cut and ligation of: |
| - | | + | |
| - | ==June, 9th== | + | |
| - | | + | |
| - | Ligation of:
| + | |
| - | | + | |
| | *I0: <partinfo>BBa_K125500</partinfo> (E-S)+<partinfo>BBa_B0015</partinfo> (E-X) | | *I0: <partinfo>BBa_K125500</partinfo> (E-S)+<partinfo>BBa_B0015</partinfo> (E-X) |
| | *I1: <partinfo>BBa_E2050</partinfo> (E-S)+<partinfo>BBa_K165018</partinfo> (E-X) | | *I1: <partinfo>BBa_E2050</partinfo> (E-S)+<partinfo>BBa_K165018</partinfo> (E-X) |
| | *I2: <partinfo>BBa_P1004</partinfo> (E-S)+<partinfo>BBa_B0015</partinfo> (E-X) | | *I2: <partinfo>BBa_P1004</partinfo> (E-S)+<partinfo>BBa_B0015</partinfo> (E-X) |
| - | *I3: <partinfo>BBa_K081008</partinfo> (E-S)+<partinfo>BBa_B0015</partinfo> (E-X) | + | *I3: <partinfo>BBa_K081008</partinfo> (E-S)+<partinfo>BBa_B0015</partinfo> (E-X)<br></td></tr> |
| - | | + | <tr><td align="center" valign="top">[[Team:UNIPV-Pavia/Calendar/June/settimana2 #June, 10th|June, 10th]]</td><td align="left">4° Bio-Lab - Transformation of ligations I0, I1, I2, I3 in home made ''E. coli'' DH5-alpha competent cells. Transformants were plated on LB+Amp agar plates.<br></td></tr> |
| - | | + | <tr><td align="center" valign="top">[[Team:UNIPV-Pavia/Calendar/June/settimana2 #June, 11th|June, 11th]]</td><td align="left">5° Bio-Lab - Inoculum from plates in liquid LB and glycerol stocks preparation for I0, I1, I2 and I3 in duplicate (2 colonies picked from each plate).<br></td></tr> |
| - | | + | <tr><td align="center" valign="top"><html><a name="June_12th"></a></html>June, 12th</td><td align="left">iGEM EU workshop.<br></td></tr> |
| - | <partinfo>BBa_B0015</partinfo> was accidentally not inoculated yesterday... :( | + | <tr><td align="center" valign="top"><html><a name="June_13th"></a></html>June, 13th</td><td align="left">iGEM EU workshop.<br></td></tr> |
| - | Luckily, we had a DNA stock for this part stored at -20°C, so we used this stock for ligations.
| + | <tr><th align="center" width="12%" colspan="2"><b>JUNE: WEEK 3</b></th></tr> |
| - | | + | <tr><td align="center" valign="top">[[Team:UNIPV-Pavia/Calendar/June/settimana3 #June, 14th|June, 14th]]</td><td align="left">6° Bio-Lab - Inoculum from glycerol stock of I0-1, I0-2, I1-1, i1-2, I2-1, I3-1 and I3-2 for screening.</td></tr> |
| - | Cultures grown overnight at 37°C 220 rpm were all saturated. BioBricks were extracted with MiniPrep kit.
| + | <tr><td align="center" valign="top">[[Team:UNIPV-Pavia/Calendar/June/settimana3 #June, 15th|June, 15th]]</td><td align="left">7° Bio-Lab - MiniPrep, gel run/cut and ligation of |
| - | Surprisingly, after the first centrifugation, we observed that <partinfo>BBa_E2050</partinfo> pellet was orange!! This is curious, because this BioBrick expresses mOrange, but it lacks of promoters. Probably, this spurious transcription is due to the promoter of Kanamycin resistance, that is oriented in the same direction.
| + | *I4: <partinfo>BBa_K165037</partinfo> (S-P) + I1 (X-P) |
| - | | + | *I5: I2 (E-S) + <partinfo>BBa_J61001</partinfo> (E-X) |
| - | <table> | + | *I6: I3 (E-S) + <partinfo> BBa_T9002</partinfo> (E-X)<br></ td></tr> |
| - | <tr> | + | <tr><td align="center" valign="top">[[Team:UNIPV-Pavia/Calendar/June/settimana3 #June, 16th|June, 16th]]</td><td align="left">8° Bio-Lab - Tranformation of I4 (DH5-alpha), I5 (TOP10) and I6 (DH5-alpha).<br></td></tr> |
| - | <td> | + | <tr><td align="center" valign="top">[[Team:UNIPV-Pavia/Calendar/June/settimana3 #June, 17th|June, 17th]]</td><td align="left">9° Bio-Lab - Colonies were picked from each plate, I4-1, I4-2, I5-1, I5-2, I6-1, I6-2 and I6-3 were grown in 1ml liquid culture and glycerol stocked (not I5-2, because no growth was observed in LB+Cm and not for I6-1, not grown). Cultures refilled with 5ml LB+antibiotic for tomorrow MiniPrep and screening.<br></td></tr> |
| - | | + | <tr><td align="center" valign="top">[[Team:UNIPV-Pavia/Calendar/June/settimana3 #June, 18th|June, 18th]]</td><td align="left">10° Bio-Lab - MiniPrep for I4-1, I4-2, I5-1, I6-2 and I6-3. Screening gel (all the clones are ok!) :)<br></td></tr> |
| - | [[Image:UNIPV_attivita_lab1.jpg|thumb|50px|left|BBa_E2050 orange pellet]] | + | <tr><th align="center" width="12%" colspan="2"><b>JUNE: WEEK 4</b></th></tr> |
| - | | + | <tr><td align="center" valign="top">[[Team:UNIPV-Pavia/Calendar/June/settimana4 #June, 21st|June, 21st]]</td><td align="left">11° Bio-Lab - Week activities planning and freezer sorting.<br></td></tr> |
| - | </td> | + | <tr><td align="center" valign="top">[[Team:UNIPV-Pavia/Calendar/June/settimana4 #June, 22nd|June, 22nd]]</td><td align="left">12° Bio-Lab - Inoculations of I6, BBa_J23118, BBa_J23110, BBa_J23114, BBa_J23116.<br></td></tr> |
| - | <td> | + | <tr><td align="center" valign="top">[[Team:UNIPV-Pavia/Calendar/June/settimana4 #June, 23rd|June, 23rd]]</td><td align="left">13° Bio-Lab - 10° Meeting - Updating about Bio-Lab activity.<br>MiniPrep of ON cultures I6, BBa_J23118, BBa_J23110, BBa_J23114, BBa_J23116, with NanoDrop quantification. Digestions, gel run and extraction of the above. We received BBa_K208001, the Silver-fusion compatible BioBrick part that codes for phasin. Streaked them on two LB+Kan agar plates (named PhaP-1 and PhaP-2)</td></tr> |
| - | After MiniPrep, purified DNA was quantified with NanoDrop.
| + | <tr><td align="center" valign="top">[[Team:UNIPV-Pavia/Calendar/June/settimana4 #June, 24th|June, 24th]]</td><td align="left">14° Bio-Lab - Colonies on both PhaP-1 and PhaP-2. Inoculation of one colony per plate, with subsequent growth for 6hrs. Glycerol stocks and refill for tomorrow MiniPrep. Yesterday's ligations heated to inactivate ligase and transformed in TOP10 homemade competent cells.<br></td></tr> |
| - | | + | <tr><td align="center" valign="top">[[Team:UNIPV-Pavia/Calendar/June/settimana4 #June, 25th|June, 25th]]</td><td align="left">15° Bio-Lab - Plates all show colonies! Picked two colonies from each plate and incubated in 1ml LB+Amp. The cultures were grown for six hours then glycerol stocks were made. PhaP-1 and 2 MiniPrep and quantification by NanoDrop. DNA quantification was poor, so we decided to perform digestion screening and to repeat Miniprep next week for sequencing. Then they were digested and gel screened, both resulting positive. <br></td></tr> |
| - | {|
| + | <tr><th align="center" width="12%" colspan="2"><b>JULY: WEEK 1</b></th></tr> |
| - | | <partinfo>BBa_E2050</partinfo> || 139,2 ng/ul
| + | <tr><td align="center" valign="top">[[Team:UNIPV-Pavia/Calendar/July/settimana1 #June, 28th|June, 28th]]</td><td align="left">16° Bio-Lab - Inoculum from glycerol stock for I7-1, I7-2, I8-1, I8-2, I9-1, I9-2, I10-1 and I10-2 in 5ml LB+Amp. Cultures grown overnight. Inoculum of PhaP-1 and PhaP-2 in 5ml LB+Kan and ON growth. Received strains and plasmid from CGSC (Yale University). Details in the week view.<br></td></tr> |
| - | |-
| + | <tr><td align="center" valign="top">[[Team:UNIPV-Pavia/Calendar/July/settimana1 #June, 29th|June, 29th]]</td><td align="left">17° Bio-Lab - From I7-1, I7-2, I8-1, I8-2, I9-1, I9-2, I10-1 and I10-2 5ul were aliquoted and diluted in 500ul LB+Amp for a preliminary TECAN test. Then cultures were MiniPrepped. Almost all cultures were digested. PhaP-1 and PhaP-2 were prepared or sequencing. All strains received from Yale Univesity were on blotting paper disks, that were placed on an LB agar plates, resuspended and then streaked. After plate streaking, disks were transferred in falcon tubes containing liquid LB.<br></td></tr> |
| - | | <partinfo>BBa_K165037</partinfo> || 181,5 ng/ul | + | <tr><td align="center" valign="top">[[Team:UNIPV-Pavia/Calendar/July/settimana1 #June, 30th|June, 30th]]</td><td align="left">18° Bio-Lab - Inoculum of cultures for a TECAN test.<br></td></tr> |
| - | |-
| + | </table> |
| - | | <partinfo> BBa_P1004</partinfo> || 132,8 ng/ ul | + | |
| - | |-
| + | |
| - | | <partinfo>BBa_K125500</partinfo> || 124,3 ng/ul | + | |
| - | |- | + | |
| - | | <partinfo>BBa_K081008</partinfo> || 138,3 ng/ul
| + | |
| - | |- | + | |
| - | | <partinfo>BBa_J61001</partinfo> || 122,2 ng/ul | + | |
| - | |- | + | |
| - | | <partinfo>BBa_K165018</partinfo> || 166,3 ng/ul | + | |
| - | |} | + | |
| | </td> | | </td> |
| | </tr> | | </tr> |
| | </table> | | </table> |
| | | | |
| - | Digestion of:
| + | <td width="15%" align="right" valign="top"> |
| - | | + | |
| - | {|
| + | |
| - | | ''Culture'' || ''Kind'' || ''Final reaction volume (ul) '' || ''DNA (ul)'' || ''H20 (ul)'' || ''Enzyme 1'' || ''Enzyme 2'' || ''Buffer H''
| + | |
| - | |-
| + | |
| - | | <partinfo>BBa_K125500</partinfo> || Insert || 25 || 16 || 4,5 || 1 EcoRI || 1 SpeI || 2,5
| + | |
| - | |-
| + | |
| - | | <partinfo>BBa_B0015</partinfo> || Vector || 25 ||6,3 || 14,2 || 1 EcoRI || 1 XbaI || 2,5
| + | |
| - | |-
| + | |
| - | | <partinfo>BBa_E2050</partinfo> || Insert || 25 || 14 || 6,5 || 1 EcoRI || 1 SpeI || 2,5
| + | |
| - | |-
| + | |
| - | | <partinfo>BBa_K165018</partinfo> || Vector || 25 ||6 || 14,5 || 1 EcoRI || 1 XbaI || 2,5
| + | |
| - | |-
| + | |
| - | | <partinfo>BBa_P1004</partinfo> || Insert || 25 || 15 || 5,5 || 1 EcoRI || 1 SpeI || 2,5
| + | |
| - | |-
| + | |
| - | | <partinfo>BBa_K081008</partinfo> || Insert || 25 || 14,5 || 6 || 1 EcoRI || 1 SpeI || 2,5
| + | |
| - | |}
| + | |
| - | | + | |
| - | Digestions were incubated at 37°C for 3 hours, gel run and gel-extracted.
| + | |
| - | <table align='center'>
| + | |
| - | <tr><td>
| + | |
| - | [[Image:UNIPV_attivita_lab3.jpg|thumb|300px|left|Gel run/cut of cultures]]
| + | |
| - | </td></tr></table>
| + | |
| - | <br>
| + | |
| - | Ligation of I0, I1, I2 and I3 was performed at 16°C overnight.
| + | |
| - | | + | |
| - | ==June, 10th== | + | |
| - | | + | |
| - | I0, I1, I2 and I3 were tranformed in home made compotent ''E. coli'' DH5-alpha and plated on LB+Amp agar plates.
| + | |
| - | | + | |
| - | Plates with transformed ''E.coli'' were incubated overnight at 37°C 220 rpm.
| + | |
| - | | + | |
| - | <table align='center'><tr><td>
| + | |
| - | [[Image:UNIPV_attivita_lab4.jpg|thumb|300px|]]
| + | |
| - | </td></tr></table>
| + | |
| - | | + | |
| - | The four plates were named:
| + | |
| - | * iGEM 2010 10/06/2010 DH5-alpha I0 LB+Amp
| + | |
| - | * iGEM 2010 10/06/2010 DH5-alpha I1 LB+Amp
| + | |
| - | * iGEM 2010 10/06/2010 DH5-alpha I2 LB+Amp
| + | |
| - | * iGEM 2010 10/06/2010 DH5-alpha I3 LB+Amp
| + | |
| - | | + | |
| - | and are stored at +4°C.
| + | |
| - | | + | |
| - | ==June, 11th==
| + | |
| - | | + | |
| - | All four plates grown overnight showed colonies!!
| + | |
| - | Two colonies for each plate were peaked and incubated in 1ml LB+Amp. For I2, a phenotipic test was performed: the same two colonies were inoculated both in LB+Amp and LB+Cm. So, 10 cultures were incubated at 37°C 220 rpm for 8 hours:
| + | |
| - | | + | |
| - | {|
| + | |
| - | | I0-1 in 1ml LB+Amp; || I0-2 in 1ml LB+Amp
| + | |
| - | |-
| + | |
| - | | I1-1 in 1ml LB+Amp; || I1-2 in 1ml LB+Amp
| + | |
| - | |-
| + | |
| - | | I2-1 in 1ml LB+Amp; || I2-2 in 1ml LB+Amp
| + | |
| - | |-
| + | |
| - | | I2-1 in 1ml LB+Cm; || I2-2 in 1ml LB+Cm
| + | |
| - | |-
| + | |
| - | | I3-1 in 1ml LB+Amp; || I3-2 in 1ml LB+Amp
| + | |
| - | |}
| + | |
| - | | + | |
| - | After over-day growth, we observed that the phenotipic test for I2 was positive for both colonies: I2-1 and I2-2 were grown both in LB+Amp and LB+Cm.
| + | |
| - | | + | |
| - | 8 glycerol stocks were prepared and stored at -80°C. Next week a screening will be performed to select positive colonies.
| + | |
| - | | + | |
| - | ==June, 14th==
| + | |
| - | | + | |
| - | Inoculum in 5ml LB+Amp from glycerol stock for:
| + | |
| - | | + | |
| - | *I0-1
| + | |
| - | *I0-2
| + | |
| - | *I1-1
| + | |
| - | *I1-2
| + | |
| - | *I2-1 (phenotipic screening ok, grown both on LB+Amp and LB+Cm)
| + | |
| - | *I3-1
| + | |
| - | *I3-2
| + | |
| - | | + | |
| - | Cultures were grown overnight at 37°C 220 rpm.
| + | |
| - | | + | |
| - | ==June, 15th==
| + | |
| - | | + | |
| - | Cultures grown overnight at 37°C 220 rpm were all saturated. BioBricks were extracted with MiniPrep kit.
| + | |
| - | | + | |
| - | After MiniPrep, purified DNA was quantified with NanoDrop.
| + | |
| - | | + | |
| - | {|
| + | |
| - | | I0-1 || 104,5 ng/ul
| + | |
| - | |-
| + | |
| - | | I0-2 || 135,2 ng/ul
| + | |
| - | |-
| + | |
| - | | I1-1 || 74 ng/ul
| + | |
| - | |-
| + | |
| - | | I1-2 || 104,3 ng/ul
| + | |
| - | |-
| + | |
| - | | I2-1 || 169,8 ng/ul
| + | |
| - | |-
| + | |
| - | | I3-1 || 256,6 ng/ul
| + | |
| - | |-
| + | |
| - | | I3-2 || 137,7 ng/ul
| + | |
| - | |}
| + | |
| - | | + | |
| - | Digestion of:
| + | |
| - | | + | |
| - | {|
| + | |
| - | | ''Culture'' || ''Kind'' || ''Final reaction volume (ul) '' || ''DNA (ul)'' || ''H20 (ul)'' || ''Enzyme 1'' || ''Enzyme 2'' || ''Buffer H''
| + | |
| - | |-
| + | |
| - | | I0-1 || Screening || 25 || 2,5 || 19 || 1 EcoRI || 1 PstI || 2,5
| + | |
| - | |-
| + | |
| - | | I0-2 || Screening || 25 ||1,9 || 18,6 || 1 EcoRI || 1 PstI || 2,5
| + | |
| - | |-
| + | |
| - | | I1-1 || Insert|| 25 || 13,5 || 7 || 1 XbaI || 1 PstI || 2,5
| + | |
| - | |-
| + | |
| - | | I1-2 || Insert|| 25 ||15 || 5,5 || 1 XbaI || 1 PstI || 2,5
| + | |
| - | |-
| + | |
| - | | I2-1 || Insert|| 25 || 11 || 9,5 || 1 EcoRI || 1 SpeI || 2,5
| + | |
| - | |-
| + | |
| - | | I3-1 || Insert|| 25 || 7,8 || 12,7 || 1 EcoRI || 1 SpeI || 2,5
| + | |
| - | |-
| + | |
| - | | I3-2 || Insert|| 25 || 14,5 || 6 || 1 EcoRI || 1 SpeI || 2,5
| + | |
| - | |-
| + | |
| - | | <partinfo>BBa_T9002</partinfo> || Vector|| 25 || 4,3 || 16,2 || 1 EcoRI || 1 XbaI || 2,5
| + | |
| - | |-
| + | |
| - | | <partinfo>BBa_K165037</partinfo> || Vector || 25 || 5,5 || 15 || 1 SpeI || 1 PstI|| 2,5
| + | |
| - | |-
| + | |
| - | | <partinfo>BBa_J61001</partinfo> || Vector || 25 || 8,2 || 12,3 || 1 EcoRI || 1 XbaI || 2,5
| + | |
| - | |}
| + | |
| - | | + | |
| - | Digestions were incubated at 37°C for 3 hours, gel run and gel-extracted.
| + | |
| - | | + | |
| - | Two gels were prepared, as shown in figure.
| + | |
| - | | + | |
| - | Gel results show that:
| + | |
| - | *I0-1 is negative, while I0-2 is positive: we choose I0-2, from now on named I0, for sequencing and for gel extraction of Insert.
| + | |
| - | *Both I1-1 and I1-2 are positive. We choose I1-2, from now on named I1, for sequencing and gel-extraction because its DNA concentration is better.
| + | |
| - | *I2-1, from now on I2, is positive so it will be used for sequencing and gel-extraction.
| + | |
| - | *I3-2 is negative, while I3-1, from now on I3, is used for sequencing and gel-extraction.
| + | |
| - | | + | |
| - | So I0-2, I1-2, I2-1 and I3-1 samples are prepared for sequencing.
| + | |
| - | | + | |
| - | Ligation of:
| + | |
| - | | + | |
| - | *I4: <partinfo>BBa_K165037</partinfo> (S-P) + I1 (X-P)
| + | |
| - | *I5: I2 (E-S) + <partinfo>BBa_J61001</partinfo> (E-X)
| + | |
| - | *I6: I3 (E-S) + <partinfo>BBa_T9002</partinfo> (E-X)
| + | |
| - | | + | |
| - | Ligation of I4, I5 and I6 was performed at 16°C overnight.
| + | |
| - | | + | |
| - | ==June, 16th==
| + | |
| - | | + | |
| - | | + | |
| - | Ligations I4, I5 and I6 were transformed in ''E. coli'':
| + | |
| - | * I4 and I6 in ''E. coli'' DH5-alpha
| + | |
| - | * I5 (final part) in ''E. coli'' TOP10
| + | |
| - | | + | |
| - | 1ul of ligation was transformed in 100ul competent cells.
| + | |
| - | | + | |
| - | ==June, 17th==
| + | |
| - | | + | |
| - | We checked the presence of colonies in plates of I4, I5 and I6 incubated overnight at 37°C.
| + | |
| - | All plates showed colonies. I4 had big, round single colonies. I5 showed big colonies surrounded by small colonies. I6 showed few small colonies and for this reason it was further incubated for 3 hours. Colonies were peaked and inoculated in 1ml LB+antibiotic.
| + | |
| - | <center>
| + | |
| - | {|
| + | |
| - | | I4-1 || LB+ Amp
| + | |
| - | |-
| + | |
| - | | I4-2 || LB+Amp
| + | |
| - | |-
| + | |
| - | | I5-1 || LB+Amp
| + | |
| - | |-
| + | |
| - | | I5-1 || LB+Cm
| + | |
| - | |-
| + | |
| - | | I5-2|| LB+Amp
| + | |
| - | |-
| + | |
| - | | I5-2 || LB+Cm
| + | |
| - | |-
| + | |
| - | | I6-1 || LB+Amp
| + | |
| - | |-
| + | |
| - | | I6-2 || LB+Amp
| + | |
| - | |-
| + | |
| - | | I6-3 || LB+Amp
| + | |
| - | |}
| + | |
| - | </center>
| + | |
| - | | + | |
| - | I5-1 and I5-2 were inoculated both in LB+Amp and LB+Cm to have a phenotipic assay: in fact I5 should express the Chloramphenicol resistance.
| + | |
| - | All 9 cultures were incubated at 37°C 220 rpm for 6 hours.
| + | |
| - | | + | |
| - | 250 ml LB and 250ml LB+Cm for low copy plasmids (91,5 ul of Cm from 1000x stock in 250 ml LB).
| + | |
| - | | + | |
| - | After six hours, we checked the growth in liquid of our colonies.
| + | |
| - | *I4-1 and I4-2 were all grown
| + | |
| - | *I5-1 and I5-2 were both grown on LB+Amp, but only I5-1 was grown in LB+Cm. I5-2 was negative at this phenotipic assay, so it was discarded.
| + | |
| - | *I6-1 was apparently not grown, so it was discarded. I6-2 and I6-2 were grown, but the cultures were more limpid than the others, showing that I6 has a slower growth than nomal.
| + | |
| - | | + | |
| - | Glycerol stocks were prepared for:
| + | |
| - | {|
| + | |
| - | | I4-1 || I4-2 || I5-1(Amp) || I6-2 || I6-3
| + | |
| - | |}
| + | |
| - | and stored at -80°C.
| + | |
| - | | + | |
| - | Remaining cultures were re-filled with 5ml LB+Amp and inoculated at 37°C 220rpm for tomorrow MiniPrep.
| + | |
| - | | + | |
| - | ==June, 18th==
| + | |
| - | | + | |
| - | All cultures incubated at 37°C 220 rpm overnight were grown. MiniPrep was performed,, with the following quantifications:
| + | |
| - | | + | |
| - | {|
| + | |
| - | | I4-1 || 218 ng/ul
| + | |
| - | |-
| + | |
| - | | I4-2 || 269 ng/ul
| + | |
| - | |-
| + | |
| - | | I5-1 || 141 ng/ul
| + | |
| - | |-
| + | |
| - | | I6-2 || 218 ng/ul
| + | |
| - | |-
| + | |
| - | | I6-3 || 169 ng/ul
| + | |
| - | |}
| + | |
| - | | + | |
| - | | + | |
| - | | + | |
| - | | + | |
| - | | + | |
| - | | + | |
| - | [[Image:I4_I5_I6_screening_unipv_18_6_10.jpg|300px|thumb|center|Gel run for screening]]
| + | |
| - | | + | |
| - | | + | |
| - | </td>
| + | |
| - | <td width="20%" valign="top">
| + | |
| - | | + | |
| - | | + | |
| | | | |
| | {{UNIPV-Pavia/menu_mesi}} | | {{UNIPV-Pavia/menu_mesi}} |
"
