From 2013.igem.org
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| | { | | { |
| - | "date" : "2013-07-05", | + | "date" : "2013-07-22", |
| - | "author" : "emil", | + | "author" : "fabio", |
| - | "title" : " Purification of the second run of Inocula of ligation and re-ligation of the digested products ", | + | "title" : " blue and red light sensors!!", |
| - | "content" : "<html> I added 1µl of DPN1 to the insert and 1µl of SAP to the backbones.Then I have purified the inocula following the <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#miniprep\"> miniprep protocol</a>.Afterwards I quantified them toghether with the results of the purification of the digestion products( performed following the <a href=\"https://2013. igem.org/Team: UNITN-Trento/Protocols#Promega-PCR-Gel\"> purification protocol < /a> ) with the following results:< /html> {{:Team:UNITN-Trento/Templates/Styles/Spoiler|Quantification|< html>< center><table class=\"tn-sp-table\"><tr><th>Sample</th><th>Quantification</th></tr><tr><td>1 :1 024+0840 E< /td> <td>482. 6ng/µl< /td> </tr><tr><td>1:1 024+0840 E</td><td>700ng/µl</td></tr><tr><td>1:1 024+0840</td><td>373. 1ng/µl</td></tr><tr><td>1:3 024+0840< /td> <td>423. 5ng/µl< /td>< /tr>< tr>< td> 1: 3 024+0840< /td> <td>327.8ng/µl</td></tr><tr><td>1:1 026+0840< /td> <td>293.1ng/µl</td></tr><tr><td>1: 1 026+0840< /td> <td>1243ng/µl</td></tr><tr><td>1: 3 026+0840< /td> <td>766.4ng/µl</td></tr><tr><td>1:3 026+0840< /td> <td>369.1ng/µl</td></tr><tr><td>GFP</td><td>15.9ng/µl</td></tr><tr><td>K823026</td><td>36.3ng/µl</td></tr><tr><td>K823026</td><td>43.7ng/µl</td></tr></table></center></html>}}<html>Then I digested 800 ng of each sample with EcoR1 HF and Pst1 following the <a href=\"https: //2013.igem.org/Team:UNITN-Trento/Protocols#Digestion\">digestion protocol< /a> with the following results:</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler| Gel order|<html><center ><table class=\"tn-sp-table\"><tr><th>Sample</th><th>Well</th></tr><tr><td>Ladder 1kb Fermentas</td><td>1</td></tr><tr><td>BBa_K823024+BBa_E0840 A</td><td>3</td></tr><tr><td>BBa_K823024+BBa_E0840 B</td><td>5</td></tr><tr><td>BBa_K823024+BBa_E0840 C</td><td>6</td></tr><tr><td>BBa_K823024+BBa_E0840 D</td><td>7</td></tr><tr><td>BBa_K823024+BBa_E0840 E</td><td>8</td></tr><tr><td>BBa_K823026+BBa_E0840 F</td><td>9</td></tr><tr><td>BBa_K823026+BBa_E0840 G</td><td>10</td></tr><tr><td>BBa_K823026+BBa_E0840 H</td><td>11</td></tr><tr><td>BBa_K823026+BBa_E0840 I</td><td>12</td></tr></table></center></html>}}{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Gel image|<html><img src=\"https://static.igem.org/mediawiki/2013/ 4/ 4b/Tn- 20130705-ET-Ligation_screening2.jpg\" width=\" 450\" /></html>}}<html> As we can see no one of the wells shows the right insert in the lower band( there is always the previous insert the RFP + Lac promoter ~ 1200 bp).At the same time I did the ligation of K823024+E0840 and K823026+E0840 previously digested following the <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#Ligation\"> ligation protocol </a>.<br>N.B. Remember always to spin the ligation buffer before using<br> After that I plated the product of the ligation as done before(1:1,1:3,ctrl for each sample) on Ampicillin added LB.</html>", | + | "content" : "<html> let’s get ready to screen!! This time I amplified my parts by pcr them out with OneTaq polymerase!! I started from the blue promoter ( number A from the firt set). Pcr sample composition: < br> 5X Onetaq standard reaction buffer 10 ul < br> 10 mM dNTPs 1 ul< br> Forward primer 1ul< br> Reverse primer 1 ul< br> One Taq 0. 25< br> Phusion 0.3< br> Template 0. 5< br> Water up to 50 ul< br> Pcr program values: < br> Initial digestion: 94° 2min < br> Digestion: 94 30 sec< br> Annealing: 60 1 min< br> Annealing: 68° 15 sec< br> Final extention: 68° 5 min< br> (30 cycles from step 3 to step 5) < br> The screening was successful : < br> </html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler| gel|<html><center><img src=\"https://static.igem.org/mediawiki/2013/ 0/ 06/Tn- 2013_C.jpg\" width=\" 450px\" / ></center></html>}}<html> Finally I purified the PCR and abtained a 209,8 ng/ul yieald.Meanwhile I started thinking about the ligation of the two blue parts: the idea is to use k592016 as plasmid and k592006 as an insert to put downstream . So I had to digest o/n 3 ug of 016(from the first set) with Spel and Pstl, and 006( the purified Pcr) with Xbal and Pstl. At the same time I digested R0010 with Ecorl and Spel, cause I’ll need to put it on top of everything once I have my biobrick.</html>", |
| - | "tags" : "K832024-K823026-E0840" | + | "tags" : " YF1_FixJ - FixK2- ho1_PcyA_cph8-OmpR" |
| | } | | } |
Revision as of 08:36, 3 October 2013
{
"date" : "2013-07-22",
"author" : "fabio",
"title" : " blue and red light sensors!!",
"content" : " let’s get ready to screen!! This time I amplified my parts by pcr them out with OneTaq polymerase!! I started from the blue promoter (number A from the firt set). Pcr sample composition:
5X Onetaq standard reaction buffer 10 ul
10 mM dNTPs 1 ul
Forward primer 1ul
Reverse primer 1 ul
One Taq 0.25
Phusion 0.3
Template 0.5
Water up to 50 ul
Pcr program values:
Initial digestion: 94° 2min
Digestion: 94 30 sec
Annealing: 60 1 min
Annealing: 68° 15 sec
Final extention: 68° 5 min
(30 cycles from step 3 to step 5)
The screening was successful :
Finally I purified the PCR and abtained a 209,8 ng/ul yieald.Meanwhile I started thinking about the ligation of the two blue parts: the idea is to use k592016 as plasmid and k592006 as an insert to put downstream . So I had to digest o/n 3 ug of 016(from the first set) with Spel and Pstl, and 006( the purified Pcr) with Xbal and Pstl. At the same time I digested R0010 with Ecorl and Spel, cause I’ll need to put it on top of everything once I have my biobrick.",
"tags" : " YF1_FixJ - FixK2- ho1_PcyA_cph8-OmpR"
}
"
![](\"https://static.igem.org/mediawiki/2013/0/06/Tn-2013_C.jpg\")
