Team:UNITN-Trento/Notebook/Labposts/06/41

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(Created page with "{ "date" : "2013-06-22", "author" : "thomas", "title" : "Minipreps and screening of AraCpBAD + EFE (in pSB1C3)", "content" : "<html>Starting from the inocula of yesterday, I ...")
 
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{
{
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"date" : "2013-06-22",
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"date" : "2013-06-20",
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"author" : "thomas",
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"author" : "thomas-viola",
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"title" : "Minipreps and screening of AraCpBAD + EFE (in pSB1C3)",
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"title" : "Cloning of AraCpBAD (in pSB1C3) + EFE (in Puc57)",
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"content" : "<html>Starting from the inocula of yesterday, I did minipreps following <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#miniprep\">this protocol</a>.</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Pellets Image|<html><center><img id=\"post_img\" src=\"https://static.igem.org/mediawiki/2013/3/36/Tn-20130622-Pellet_TP.jpg\" width=\"450px\" /></center></html>}}<html></html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Quantification results|<html><center><table class=\"tn-sp-table\"><tr><th>Sample</th><th>ng/ul</th></tr><tr><td>1:1 Plate1 n°1</td><td>1201,5</td></tr><tr><td>1:1 Plate1 n°2</td><td>1620,5</td></tr><tr><td>1:2 Plate1 n°1</td><td>969,8</td></tr><tr><td>1:4 Plate1 n°1</td><td>636,3</td></tr><tr><td>1:1 Plate2 n°1</td><td>672,8</td></tr><tr><td>1:1 Plate2 n°2</td><td>1222,5</td></tr><tr><td>1:2 Plate2 n°1</td><td>782,0</td></tr><tr><td>1:4 Plate2 n°1</td><td>705,3</td></tr></table></center></html>}}<html>As you can see we obtained a set of very high concentrations. We proceeded then with the screening test. To do that we digested 800 ng of DNA with EcorI and PstI following <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#Digestion\">the screening digestion protocol</a>. In the end we prepared the sample for an electrophoresis analysis.</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Gel Image|<html><img id=\"post_img\" src=\"https://static.igem.org/mediawiki/2013/8/8b/Tn-20130622-Thomas_AraCpBAD_EFE.jpg\" width=\"450px\"/></html>}}<html> <br/> As you can see, 6 samples out of 8 have the expected bands: 2070bp for the vector and 2300bp for AraCpBAD.</html>",
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"content" : "<html>We started digesting 500 ng of AraCpBAD in pSB1C3 with the SpeI and PstI and 500 ng of EFE in Puc57 with XbaI and PstI following <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#Digestion\">digestion for Biobricks protocol</a>.<br/><br/>For the digested vector (AraCpBAD), we incubated the part for one hour at 37&deg;C with the SAP phosphatase before disactivating the enzymes.<br/><br/>We then preceeded by ligating and transforming the two parts in competent NEB10b cells following <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#Digestion\">the ligation protocol for Biobricks </a>.<br/>We decided to do that in duplicate and with different ratios plasmid:insert (ctrl, 1:1, 1:2, 1:4) obtaining so 8 reactions.<br/><b>Results:</b> as you can see from the pictures, we there are only few colonies in the control so we hope our construct is correctly cloned. The next step will be the inocula and the screening test.</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|AraCpBAD (in pSB1C3) + EFE (in Puc57) Ctrl|<html><center><img id=\"post_img\" src=\"https://static.igem.org/mediawiki/2013/5/5f/Tn-20130620-Puc57_Ctrl.jpg\" width=\"400px\"/></center></html>}}{{:Team:UNITN-Trento/Templates/Styles/Spoiler|AraCpBAD (in pSB1C3) + EFE (in Puc57) 1:1|<html><center><img id=\"post_img\" src=\"https://static.igem.org/mediawiki/2013/f/f5/Tn-20130620-Puc57_1-1.jpg\" width=\"400px\"/></center></html>}}{{:Team:UNITN-Trento/Templates/Styles/Spoiler|AraCpBAD (in pSB1C3) + EFE (in Puc57) 1:2|<html><center><img id=\"post_img\" src=\"https://static.igem.org/mediawiki/2013/5/5e/Tn-20130620-Puc57_1-2.jpg\" width=\"400px\"/></center></html>}}{{:Team:UNITN-Trento/Templates/Styles/Spoiler|AraCpBAD (in pSB1C3) + EFE (in Puc57) 1:4|<html><center><img id=\"post_img\" src=\"https://static.igem.org/mediawiki/2013/a/ab/Tn-20130621-Puc57_1-4.jpg\" width=\"400px\"/></center></html>}}",
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"tags" : "EFE-AraCpBAD"
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"tags" : "AraCpBAD-EFE"
}
}

Latest revision as of 07:51, 3 October 2013

{ "date" : "2013-06-20", "author" : "thomas-viola", "title" : "Cloning of AraCpBAD (in pSB1C3) + EFE (in Puc57)", "content" : "We started digesting 500 ng of AraCpBAD in pSB1C3 with the SpeI and PstI and 500 ng of EFE in Puc57 with XbaI and PstI following digestion for Biobricks protocol.

For the digested vector (AraCpBAD), we incubated the part for one hour at 37°C with the SAP phosphatase before disactivating the enzymes.

We then preceeded by ligating and transforming the two parts in competent NEB10b cells following the ligation protocol for Biobricks .
We decided to do that in duplicate and with different ratios plasmid:insert (ctrl, 1:1, 1:2, 1:4) obtaining so 8 reactions.
Results: as you can see from the pictures, we there are only few colonies in the control so we hope our construct is correctly cloned. The next step will be the inocula and the screening test.

AraCpBAD (in pSB1C3) + EFE (in Puc57) Ctrl
AraCpBAD (in pSB1C3) + EFE (in Puc57) 1:1
AraCpBAD (in pSB1C3) + EFE (in Puc57) 1:2
AraCpBAD (in pSB1C3) + EFE (in Puc57) 1:4
", "tags" : "AraCpBAD-EFE" }

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