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

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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"}

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+{"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" : "2013-07-02",
-	"author" : "emil",
-	"title" : "Purification of the digestion products (K823026, K823024, E0840) ligation of the GFP in the 2 backbones and transformation of the ligation products ",
-	"content" : "<html>This morning I added 1&micro;l of DPN1 to the GFP ligation and 1&micro;l of SAP(alkaline phosphatase) to the 2 backbones.After 1h 30 min I have stopped the reaction by putting the reaction tubes at 80 C&deg;.Afterwards I have purified the products following the <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#Promega-PCR-Gel\"> purification protocol</a>.Then I quantified the products with the following results:</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Quantification|<html><table>     <tr><th>Sample</th><th>Quantification</th></tr><tr><td>BBa_E0840</td><td> 15.8ng/&micro;l</td></tr><tr><td>BBa_K823026</td><td> 37.6ng/&micro;l</td></tr><tr><td>BBa_K823024</td><td> 36.4ng/&micro;l</td></tr></table></html>}}<html>Then I performed the ligation(1:1,1:3,CTRL) of the GFP with the 2 backbone individually and following the <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#Ligation\"> ligation protocol</a>.Finally I transformed 10&micro;l of the ligation protocol in Neb10&beta; and plate them on ampicillin LB agar following the <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#Competent-cells-transformation\"> transformation protocol</a>.</html>",
-	"tags" : "E0840-K823026-K823024"
-}