Team:UNITN-Trento/Notebook/Labposts/07/36

From 2013.igem.org

(Difference between revisions)

Latest revision as of 10:40, 3 October 2013

{"date" : "2013-07-17","author" : "gabriele-caterina","title" : "A short day","content" : "

Screening with AgeI

First, Caterina performed a screening using AgeI on the \"1:1 A\" sample from 15/07.

	11A
template	4.34µl
AgeI-HF	1µl
EcoRI-HF
NEBuffer4	2µl
BAS
Water	9.66µl

But then we realized the Gabriele was mistaken, AgeI is able to cut both Plac+RFP and SAMsynthetase so it is not the correct enzyme to perform the screening with...

Short digestion

Gabriele then performed a short digestion (aka: a digestion similar to the screening run for 2 hours) with the sample G2_EX-SAMsynth-SP (62.6ng/µl from 15/07) and G4A_linear-pSB1C3 (41.9ng/µl from 01/07).

Digestion mixes

	EX-SAM-SP	linear-pSB1C3
template	24µl	23.87µl
EcoRI-HF	1µl
PstI
NEBuffer2	3µl
BSA
water	3µl	3.13µl

After the 2 hours of incubation, Gabriele added 1µl of DpnI to the G2 sample and 1µl of SAP to the G4A sample and incubated them at 37°C for 1h.

Digestions purification

After that, Gabriele purificated both today's short digestion and the O/N digestion from yesterday.

Sample	Digestion	Quantity
EX-SAMsynth-SP	Short	39.0ng/µl
linear-pSB1C3	Short	12.1ng/µl
EX-SAMsynth-SP	O/N	13.1ng/µl
linear-pSB1C3	O/N	7.8ng/µl

Short digest Ligation

So, Gabriele performed a ligation of the short digested samples.

	Ctrl	1:1	1:2	1:3
buffer	3.5µl
plasmid	5µl
insert	0	8µl	16µl	26µl
Ligase	1µl
Water	25.5µl	17.5µl	9.5µl	0

Then the ligation mixes were incubated at room temperature for 2 hours.

O/N digest Ligation

Finally, Gabriele performed a ligation of the overnight digested samples.

	Ctrl	1:1
Buffer	3.5µl
Plasmid	15µl
Insert	0	15µl
Ligase	1µl
Water	15.5µl	0.5µl

Then, the ligation mixes were incubated at room temperature for 2 hourse.","tags" : "SAMsynthetase"}

@@ Line 1: / Line 1: @@
-{
+{"date" : "2013-07-17","author" : "gabriele-caterina","title" : "A short day","content" : "<html><h3>Screening with AgeI</h3>First, Caterina performed a screening using AgeI on the \"1:1 A\" sample from <a href=\"https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook#tn-post-2013-07-15-gabriele\">15/07</a>.</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Digestion mix|<html><center><table class=\"tn-sp-table\"><tr><td style=\"border:none;\"></td><th>11A</th></tr><tr><td>template</td><td>4.34&micro;l</td></tr><tr><td>AgeI-HF</td><td rowspan=\"2\">1&micro;l</td></tr><tr><td>EcoRI-HF</td></tr><tr><td>NEBuffer4</td><td rowspan=\"2\">2&micro;l</td></tr><tr><td>BAS</td></tr><tr><td>Water</td><td>9.66&micro;l</td></tr></table></center></html>}}<html>But then we realized the Gabriele was mistaken, AgeI is able to cut both Plac+RFP and SAMsynthetase so it is not the correct enzyme to perform the screening with...<br><hr><h3>Short digestion</h3>Gabriele then performed a short digestion (aka: a digestion similar to the screening run for 2 hours) with the sample G2_EX-SAMsynth-SP (62.6ng/&micro;l from <a href=\"https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook#tn-post-2013-07-15-gabriele\">15/07</a>) and G4A_linear-pSB1C3 (41.9ng/&micro;l from <a href=\"https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook#tn-post-2013-07-01-Gabriele\">01/07</a>).</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Digestion mixes|<html><center><table class=\"tn-sp-table\"><tr><td style=\"border:none;\"></td><th>EX-SAM-SP</th><th>linear-pSB1C3</th></tr><tr><td>template</td><td>24&micro;l</td><td>23.87&micro;l</td></tr><tr><td>EcoRI-HF</td><td rowspan=\"2\" colspan=\"2\">1&micro;l</td></tr><tr><td>PstI</td></tr><tr><td>NEBuffer2</td><td rowspan=\"2\" colspan=\"2\">3&micro;l</td></tr><tr><td>BSA</td></tr><tr><td>water</td><td>3&micro;l</td><td>3.13&micro;l</td></tr></table></center></html>}}<html>After the 2 hours of incubation, Gabriele added 1&micro;l of DpnI to the G2 sample and 1&micro;l of SAP to the G4A sample and incubated them at 37&deg;C for 1h.<br><hr><h3>Digestions purification</h3>After that, Gabriele purificated both today's short digestion and the O/N digestion from <a href=\"https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook#tn-post-2013-07-17-gabriele-caterina\">yesterday</a>.<center><table class=\"tn-sp-table\"><tr><th>Sample</th><th>Digestion</th><th>Quantity</th></tr><tr><td>EX-SAMsynth-SP</td><td>Short</td><td>39.0ng/&micro;l</td></tr><tr><td>linear-pSB1C3</td><td>Short</td><td>12.1ng/&micro;l</td></tr><tr><td>EX-SAMsynth-SP</td><td>O/N</td><td>13.1ng/&micro;l</td></tr><tr><td>linear-pSB1C3</td><td>O/N</td><td>7.8ng/&micro;l</td></tr></table></center><br><hr><h3>Short digest Ligation</h3>So, Gabriele performed a ligation of the short digested samples.<center><table class=\"tn-sp-table\"><tr><td style=\"border:none;\"></td><th>Ctrl</th><th>1:1</th><th>1:2</th><th>1:3</th></tr><tr><td>buffer</td><td colspan=\"4\">3.5&micro;l</td></tr><tr><td>plasmid</td><td colspan=\"4\">5&micro;l</td></tr><tr><td>insert</td><td>0</td><td>8&micro;l</td><td>16&micro;l</td><td>26&micro;l</td></tr><tr><td>Ligase</td><td colspan=\"4\">1&micro;l</td></tr><tr><td>Water</td><td>25.5&micro;l</td><td>17.5&micro;l</td><td>9.5&micro;l</td><td>0</td></tr></table></center>Then the ligation mixes were incubated at room temperature for 2 hours.<br><hr><h3>O/N digest Ligation</h3>Finally, Gabriele performed a ligation of the overnight digested samples.<center><table class=\"tn-sp-table\"><tr><td style=\"border:none;\"></td><th>Ctrl</th><th>1:1</th></tr><tr><td>Buffer</td><td colspan=\"2\">3.5&micro;l</td></tr><tr><td>Plasmid</td><td colspan=\"2\">15&micro;l</td></tr><tr><td>Insert</td><td>0</td><td>15&micro;l</td></tr><tr><td>Ligase</td><td colspan=\"2\">1&micro;l</td></tr><tr><td>Water</td><td>15.5&micro;l</td><td>0.5&micro;l</td></tr></table></center>Then, the ligation mixes were incubated at room temperature for 2 hourse.</html>","tags" : "SAMsynthetase"}
-	"date" : "2013-07-19",
-	"author" : "caterina",
-	"title" : "SAM sythetase and other miniprep",
-	"content" : "<html>Yesterday, I performed inocula from different plates prepared by Gire. After miniprep and screening we had the confirmation that into pSB1C3 there was RFP and not SAM synthetase. So on Monday we will repeat the experiment.<br/><br/>Then I performed the miniprep of some inocula that I prepared the yesterday containing K1065101 and K1065102.</html>",
-	"tags" : "SAM-K1065101-K1065102"
-}