Team:UNITN-Trento/Notebook/Labposts/06/55

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Latest revision as of 07:56, 3 October 2013

{ "date" : "2013-06-25", "author" : "emil", "title" : "Purification and digestion of R0010", "content" : "I purified, and then quantified, 3 out of 5 of the previous inocula following the miniprep protocol with vacuum.

Quantification results

Sample	Quantity
1	396.1ng/µl
2	523.1ng/µl
3	171ng/µl

Afterwards, I digested 800ng of each sample with Spe1-HF and Pst1-HF following the digestion protocol for screening. Finally I ran the products on a gel (1,5% agarose, due to the short length of the insert: only 200 bp + prefix and suffix). I loaded 12µl of a ladder 100bp prepared as follows:

Ladder

	Quantity
Water	4µl
Sharpmass Euroclone 100bp ladder	1µl
glicerol solution(30%)	1µl

This time, I loaded 12µl (10µl of sample and 2µl of glicerol solution 30%) of each sample.

Gel order

Sample	Well
2µl loading dye	1
Ladder	2
sample 1	3
sample 2	4
sample 3	5
sample 4(Bruno e Fabio)	6
sample 4(Bruno e Fabio)	6
Ladder 1000 kb	7
loading dye	8

Gel Image

We can see 2 bands: the highest is the backbone (pSB1A2: 2079bp) and the lower is pLac (nearly 243bp, located between the bands 300 and 200bp of the ladder).
After the screening I digested 3µg of the pSB1A2+R0010 (sample #2) with SpeI and PstI-HF o/n following the usual protocol.", "tags" : "Plac" }

@@ Line 1: / Line 1: @@
 {
-	"date" : "2013-06-26",
+	"date" : "2013-06-25",
-	"author" : "caterina-michele",
+	"author" : "emil",
-	"title" : "Don't Trust few colonies!!!",
+	"title" : "Purification and digestion of R0010",
-	"content" : " The results of the transformations were not so good. We've only few colonies on each plate (only on Michele's control there were many colonies!). Despite this, we've decided to do inocula of some colonies in the afternoon. On the rest of the day Michele tried two different PCRs to obtain pSB1C3 linearized (someone stole his supplies!!). Obviously no results (immagini gel?). Caterina repeated the digstion but she used two differents type of ligase for the reaction of ligation. At the end of the day she transformed Neb&alpha; with these new products of ligation.   ",
+	"content" : "<html>I purified, and then quantified, 3 out of 5 of the previous inocula following the <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#miniprep\">miniprep protocol with vacuum</a>.</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Quantification results|<html><center><table class=\"tn-sp-table\"><tr><th>Sample</th><th>Quantity</th></tr><tr><td>1</td><td>396.1ng/&micro;l</td></tr><tr><td>2</td><td>523.1ng/&micro;l</td></tr><tr><td>3</td><td>171ng/&micro;l</td></tr></table></center></html>}}<html>Afterwards, I digested 800ng of each sample  with Spe1-HF and Pst1-HF following the <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#Digestion\">digestion protocol for screening</a>. Finally I ran the products on a gel (1,5% agarose, due to the short length of the insert: only 200 bp + prefix and suffix). I loaded 12&micro;l of a ladder 100bp prepared as follows:</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Ladder|<html><center><table class=\"tn-sp-table\"><tr><td style=\"border: none;\"></td><th>Quantity</th></tr><tr><td>Water</td><td>4&micro;l</td></tr><tr><td>Sharpmass Euroclone 100bp ladder </td><td>1&micro;l</td></tr><tr><td>glicerol solution(30%)</td><td>1&micro;l</td></tr></table></center></html>}}<html>This time, I loaded 12&micro;l (10&micro;l of sample and 2&micro;l of glicerol solution 30%) of each sample.</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>2&micro;l loading dye</td><td>1</td></tr><tr><td>Ladder</td><td>2</td></tr><tr><td>sample 1</td><td>3</td></tr><tr><td>sample 2</td><td>4</td></tr><tr><td>sample 3</td><td>5</td></tr><tr><td>sample 4(Bruno e Fabio)</td><td>6</td></tr><tr><td>sample 4(Bruno e Fabio)</td><td>6</td></tr><tr><td>Ladder 1000 kb</td><td>7</td></tr><tr><td>loading dye</td><td>8</td></tr></table></center></html>}}{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Gel Image|<html><img src=\"https://static.igem.org/mediawiki/2013/b/b4/Tn-20130625-EG_FB_20min.jpg\" width=\"450px\"></html>}}<html>We can see 2 bands: the highest is the backbone (pSB1A2: 2079bp) and the lower is pLac (nearly 243bp, located between the bands 300 and 200bp of the ladder).<br/>After the screening I digested 3&micro;g of the pSB1A2+R0010 (sample #2) with SpeI and PstI-HF o/n following the usual protocol.</html>",
-	"tags" : "PchBA-BMST1-araC-pBAD"
+	"tags" : "Plac"
 }