Team:UNITN-Trento/Notebook/Labposts/07/28

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Latest revision as of 10:38, 3 October 2013

{"date" : "2013-07-11","author" : "gabriele","title" : "Extraction (again) and ligation","content" : "

SAM synthetase extraction

Last time I determined the correct protocol to extract SAM synthetase from the E. coli using the new plasmid (at the end, the protocol was the usual one). I performed three PCRs (G1, G2, G3) and run the products on 1% agarose gel.

Gel

Loading scheme
1kb ladder	G1	G2	G3

$\"Gel\"$

Ligation

At first, I added 1µl of SAP to pSB1C3 O/N digestion and 1µl of DpnI to SAMsynthetase O/N digestion, and incubated the samples at 37°C for 1.5h. Then I quantified the digestions.

Sample	Quantity
SAM synthetase	12.2ng/µl
pSB1C3	14.0ng/µl

Then I performed the ligation and left the reaction run for 2h at room temperature.

Ligation Mixes

	CTRL	1:1	1:2
Buffer	4µl
Plasmid	14.29µl
Insert	0	9.15µl	18.3µl
Ligase	1µl
Water	20.71µl	11.56µl	2.41µl

Then I transformed the ligation products in NEB10β","tags" : "SAMsynthetase"}

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+{"date" : "2013-07-11","author" : "gabriele","title" : "Extraction (again) and ligation","content" : "<html><h3>SAM synthetase extraction</h3><a href=\"https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook#tn-post-2013-07-10-gabriele-viola\">Last time</a> I determined the correct protocol to extract SAM synthetase from the <i>E. coli</i> using the new plasmid (at the end, the protocol was <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#SAM-extraction-genome-coli\">the usual one</a>). I performed three PCRs (G1, G2, G3) and run the products on 1% agarose gel.</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Gel|<html><center><table class=\"tn-sp-table\"><tr><th colspan=\"4\">Loading scheme</th></tr><tr><td>1kb ladder</td><td>G1</td><td>G2</td><td>G3</td></tr></table><img src=\"https://static.igem.org/mediawiki/2013/f/f4/Tn-20130711-GG_SAMsynthetase_extraction_EXSP.jpg\" alt=\"Gel\" title=\"Gel\" width=\"450px\" /></center></html>}}<html><br><hr><h3>Ligation</h3>At first, I added 1&micro;l of SAP to pSB1C3 O/N digestion and 1&micro;l of DpnI to SAMsynthetase O/N digestion, and incubated the samples at 37&deg;C for 1.5h. Then I quantified the digestions.<center><table class=\"tn-sp-table\"><tr><th>Sample</th><th>Quantity</th></tr><tr><td>SAM synthetase</td><td>12.2ng/&micro;l</td></tr><tr><td>pSB1C3</td><th>14.0ng/&micro;l</th></tr></table></center><br/>Then I performed the ligation and left the reaction run for 2h at room temperature.</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Ligation Mixes|<html><center><table class=\"tn-sp-table\"><tr><td style=\"border:none;\"></td><th>CTRL</th><th>1:1</th><th>1:2</th></tr><tr><td>Buffer</td><td colspan=\"3\">4&micro;l</td></tr><tr><td>Plasmid</td><td colspan=\"3\">14.29&micro;l</td></tr><tr><td>Insert</td><td>0</td><td>9.15&micro;l</td><td>18.3&micro;l</td></tr><tr><td>Ligase</td><td colspan=\"3\">1&micro;l</td></tr><tr><td>Water</td><td>20.71&micro;l</td><td>11.56&micro;l</td><td>2.41&micro;l</td></tr></table></center></html>}}<html>Then I transformed the ligation products in NEB10&beta;</html>","tags" : "SAMsynthetase"}
-	"date" : "2013-07-30",
-	"author" : "thomas",
-	"title" : "Bacillus transformation!!!",
-	"content" : "<html>In order to test the transformation protocol for <i>B. subtilis</i> that Groeningen team kindly sent to us I prepared a competent cell Media following <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#subtilis-transformation\">this protocol</a>. I proceeded then perfonming the transformation using the empty vector BBa_K823026. I finally plated the tranformed cells into LB agar plates with 10 ug/ml of Kanamycin. The day after took a picture of the plate and performed a Gram staining following <a href=\"\">this protocol</a>. </html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Plate image|<html><center><img src=\"https://static.igem.org/mediawiki/2013/e/ec/Tn-2013_bacillus_transformation_plate_1.jpg\" style =\"width: 450px\"></center></html>}}{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Bacillus microscope image|<html><center><img src=\"https://static.igem.org/mediawiki/2013/6/65/Tn-2013_gram_staining_bacillus.jpg\" style =\"width: 450px\"></center></html>}}<html> As you can see from the pictures, seems that <i>Bacillus</i> was successful transformed!!!!</html>",
-	"tags" : "b. subtilis-pspac"
-}