Team:UNITN-Trento/Notebook/Labposts/07/02

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Latest revision as of 10:31, 3 October 2013

{"date" : "2013-07-01","author" : "Gabriele","title" : "Too many things in one single day","content" : "

(1) PCR: SAM synthetase amplification

I amplified SAM synthetase by performing a PCR on the samples G1 and E2 from 17/06.

Sample	Quantification
G1	80ng/µl
E2	60ng/µl

The PCR was performed following the usual SAM extraction protocol.

PCR mixes

	G1 mix	E2 mix
Template(50ng)	0.63µl	0.83µl
dNTPs	0.5µl
primer Fw	1µl
primer Rv	1µl
buffer RBC	5µl
Phusion pol	0.3µl
RBC pol	0.25µl
water	41.32µl	41.12µl

PCR results were then run on a 1% agarose gel:

Gel

Loading scheme
1kb ladder	G1	E2

$\"Gel\"$

Since the gel shows two bands at nearly 1200bp, the PCR results are confirmed. The PCR products were then purified with the Promega kit.

Sample	Type	Quantity
G1	SAM synthetase	35.6ng/µl
E2	SAM synthetase	31.5ng/µl

(2) pSB1A2+R0010+SAMsynthetase ligation screening

Then I screened the inocula from the 26/06 ligation. First I miniprepped the inocula:

Miniprep quantities

1:1	A	490.9ng/µl
B	252.1ng/µl
C	345.6ng/µl
D	295.3ng/µl
E	315.6ng/µl
F	226.2ng/µl
1:3	A	345.7ng/µl
B	147.0ng/µl
C	268.0ng/µl

The samples were then digested using the screening digestion protocol and then run on a 1% agarose gel.

Gel

Loading scheme
1kb ladder
1:1 A
1:1 B
1:1 C
1:1 D
1:1 E
1:1 F
1kb ladder
1:3 A
1:3 B
1:3 C

$\"gel\"$

Given that the gel shows only two bands (one at nearly 2kbp and the other at 200bp), SAM synthetase is not present and the ligation failed.

(3) Linear pSB1C3 purification

I also purified with the usual protocol the linearized pSB1C3 from 19/06.

	Sample	Type	Quantity
G2	linear pSB1C3	74.4ng/µl
G3	linear pSB1C3	63.3ng/µl
G2A	linear pSB1C3	44.0ng/µl
G3A	linear pSB1C3	44.8ng/µl
G4A	linear pSB1C3	41.9ng/µl

(4) O/N Digestion

Finally, I prepared an overnight digestion of the samples G1 (SAM synthetase) and G2 (linear pSB1C3) to try again the ligation tomorrow. I followed the usual protocol.

Digestion mix

	G1 SAMsynthetase	G2 linear pSB1C3
template (3µg)	48.5µl	40µl
XbaI	2.5µl	1.5µl
PstI	2.5µl	1.5µl
NEBuffer 2	10µl	5µl
water	26.5µl	0

","tags" : "SAMsynthetase-Plac-pSB1A2-pSB1C3"}

@@ Line 1: / Line 1: @@
-{
+{"date" : "2013-07-01","author" : "Gabriele","title" : "Too many things in one single day","content" : "<html><h3>(1) PCR: SAM synthetase amplification</h3>I amplified SAM synthetase by performing a PCR on the samples <b>G1</b> and <b>E2</b> from <a href=\"https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook#tn-post-2013-06-17-gabriele-emil\">17/06</a>.<center><table class=\"tn-sp-table\"><tr><th>Sample</th><th>Quantification</th></tr><tr><td>G1</td><td>80ng/&micro;l</td></tr><tr><td>E2</td><td>60ng/&micro;l</td></tr></table></center><br/>The PCR was performed following the usual <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#SAM-extraction-genome-coli\">SAM extraction protocol</a>.<br/></html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|PCR mixes|<html><center><table class=\"tn-sp-table\"><tr><td style=\"border:none;\"></td><th>G1 mix</th><th>E2 mix</th></tr><tr><td>Template(50ng)</td><td>0.63&micro;l</td><td>0.83&micro;l</td></tr><tr><td>dNTPs</td><td colspan=\"2\">0.5&micro;l</td></tr><tr><td>primer Fw</td><td colspan=\"2\">1&micro;l</td></tr><tr><td>primer Rv</td><td colspan=\"2\">1&micro;l</td></tr><tr><td>buffer RBC</td><td colspan=\"2\">5&micro;l</td></tr><tr><td>Phusion pol</td><td colspan=\"2\">0.3&micro;l</td></tr><tr><td>RBC pol</td><td colspan=\"2\">0.25&micro;l</td></tr><tr><td>water</td><td>41.32&micro;l</td><td>41.12&micro;l</td></tr></table></center></html>}}<html><br/>PCR results were then run on a 1% agarose gel:<br/></html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Gel|<html><center><table class=\"tn-sp-table\"><tr><th colspan=\"3\">Loading scheme</th></tr><tr><td>1kb ladder</td><td>G1</td><td>E2</td></tr></table><img src=\"https://static.igem.org/mediawiki/2013/8/81/Tn-20130701-GGET_GFPpcr_SAMampli.jpg\" alt=\"Gel\" width=\"450px\"><br/></center></html>}}<html><br/>Since the gel shows two bands at nearly 1200bp, the PCR results are confirmed. The PCR products were then purified with the <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#Promega-PCR-Gel\">Promega kit</a>.<center><table class=\"tn-sp-table\"><tr><th>Sample</th><th>Type</th><th>Quantity</th></tr><tr><td>G1</td><td>SAM synthetase</td><td>35.6ng/&micro;l</td></tr><tr><td>E2</td><td>SAM synthetase</td><td>31.5ng/&micro;l</td></tr></table></center><br/><hr><h3>(2) pSB1A2+R0010+SAMsynthetase ligation screening</h3>Then I screened the inocula from the <a href=\"https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook#tn-post-2013-06-26-emil-gabriele\">26/06</a> ligation. First I miniprepped the inocula:<br/></html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Miniprep quantities|<html><center><table class=\"tn-sp-table\"><tr><td rowspan=\"6\">1:1</td><td>A</td><td>490.9ng/&micro;l</td></tr><tr><td>B</td><td>252.1ng/&micro;l</td></tr><tr><td>C</td><td>345.6ng/&micro;l</td></tr><tr><td>D</td><td>295.3ng/&micro;l</td></tr><tr><td>E</td><td>315.6ng/&micro;l</td></tr><tr><td>F</td><td>226.2ng/&micro;l</td></tr><tr><td rowspan=\"3\">1:3</td><td>A</td><td>345.7ng/&micro;l</td></tr><tr><td>B</td><td>147.0ng/&micro;l</td></tr><tr><td>C</td><td>268.0ng/&micro;l</td></tr></table></center></html>}}<html><br/>The samples were then digested using the <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#Digestion\">screening digestion protocol</a> and then run on a 1% agarose gel.<br/></html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Gel|<html><center><table class=\"tn-sp-table\"><tr><th>Loading scheme</th></tr><tr><td>1kb ladder</td></tr><tr><td>1:1 A</td></tr><tr><td>1:1 B</td></tr><tr><td>1:1 C</td></tr><tr><td>1:1 D</td></tr><tr><td>1:1 E</td></tr><tr><td>1:1 F</td></tr><tr><td>1kb ladder</td></tr><tr><td>1:3 A</td></tr><tr><td>1:3 B</td></tr><tr><td>1:3 C</td></tr></table><img src=\"https://static.igem.org/mediawiki/2013/e/ef/Tn-20130701-GGET_GFP_PSB1A2R0010SAMsynth.jpg\" alt=\"gel\" width=\"450px\" /></center></html>}}<html><br/>Given that the gel shows only two bands (one at nearly 2kbp and the other at 200bp), SAM synthetase is not present and the ligation failed.<br/><br/><hr><h3>(3) Linear pSB1C3 purification</h3>I also purified with <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#Promega-PCR-Gel\">the usual protocol</a> the linearized pSB1C3 from <a href=\"https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook#tn-post-2013-06-19-gabriele-emil-bruno\">19/06</a>.<br/><center><table class=\"tn-sp-table\"><tr><td style=\"border:none;\"></td><th>Sample</th><th>Type</th><th>Quantity</th></tr><tr><td>G2</td><td>linear pSB1C3</td><td>74.4ng/&micro;l</td></tr><tr><td>G3</td><td>linear pSB1C3</td><td>63.3ng/&micro;l</td></tr><tr><td>G2A</td><td>linear pSB1C3</td><td>44.0ng/&micro;l</td></tr><tr><td>G3A</td><td>linear pSB1C3</td><td>44.8ng/&micro;l</td></tr><tr><td>G4A</td><td>linear pSB1C3</td><td>41.9ng/&micro;l</td></tr></table></center><br/><hr><h3>(4) O/N Digestion</h3>Finally, I prepared an overnight digestion of the samples <b>G1</b> (<i>SAM synthetase</i>) and <b>G2</b> (<i>linear pSB1C3</i>) to try again the ligation tomorrow. I followed the <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#Digestion\">usual protocol</a>.</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Digestion mix|<html><center><table class=\"tn-sp-table\"><tr><td style=\"border:none;\"></td><th>G1 SAMsynthetase</th><th>G2 linear pSB1C3</th></tr><tr><td>template (3&micro;g)</td><td>48.5&micro;l</td><td>40&micro;l</td></tr><tr><td>XbaI</td><td>2.5&micro;l</td><td>1.5&micro;l</td></tr><tr><td>PstI</td><td>2.5&micro;l</td><td>1.5&micro;l</td></tr><tr><td>NEBuffer 2</td><td>10&micro;l</td><td>5&micro;l</td></tr><tr><td>water</td><td>26.5&micro;l</td><td>0</td></tr></table></center></html>}}<html></html>","tags" : "SAMsynthetase-Plac-pSB1A2-pSB1C3"}
-	"date" : "2013-07-23",
-	"author" : "caterina",
-	"title" : "Trasformation of SAM",
-	"content" : "<html>I performed the ligation and the trasformation of SAMsynthetase in pSB1C3 with ROO10.<br/><br/>Also I transformed Gabriele's ligations from <a href=\"https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook#tn-post-2013-07-17-gabriele-caterina\">17/07</a> into NEB10&beta;.</html>",
-	"tags" : "R0010-SAMsynthetase"
-}