Team:UNITN-Trento/Notebook/Labposts/07/04

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Latest revision as of 10:32, 3 October 2013

{"date" : "2013-07-02","author" : "gabriele","title" : "Another busy day","content" : "

SAM synthetase ligation in (linear) pSB1C3

First of all, early in the morning I added 1µl of DpnI to the SAMsynthetase#G2 O/N digestion and 1µl of SAP to the linear_pSB1C3#G2 digestion and then incubated them at 37°C for 1.5 hours (afterward I inhibited the enzyme with 20 minutes at 80°C).

Then, I purified and quantified the two digestion samples:

Sample	Quantity
G1 SAMsynthetase	18.8ng/µl
G2 linear pSB1C3	6.5ng/µl

After that, I performed the ligation (the plasmid had a too low concentration).

	Ctrl	0.5:1	0.5:2
Buffer	3µl
Plasmid	14.3µl
Insert	0	6µl	12µl
Ligase	1µl
Water	11.7µl	5.7µl	0µl

And, \"finally\", I transformed the ligations in NEB10β cells.

(2) SAMsynthetase amplification

I amplified SAMsynthetase from the E2 sample (that was purified yesterday) following the usual protocol. The PCR was performed in triplicates.

The PCR products were run on a 1% agarose gel.

Gel

Loading scheme
E1A	E1B	E1C	empty	1kb ladder

$\"Gel\"$

As shown in the gel, a band at nearly 1200bp is present, confirming the success of the PCR.

(3) R0010 amplification

I also amplified R0010 from A sample (R0010, 243.7ng/µl) from 27/06. Being the first time amplifying this sequence, I performed (in triplicates or duplicates) a Phusion PCR using as primers the prefix Fw (Tm = 86°C) and the suffix Rv (Tm = 90°C).

PCR Mixes

	Mix HF (x3)	Mix GC (x2)
Phusion GC buffer	0	10µl
Phusion HF buffer	10µl	0
dNTPs	1µl
primer Fw	2.5µl
primer Rv	2.5µl
template (50ng/µl)	0.5µl
Phusion pol	0.5µl
Water	33µl

Given that R0010 is 200bp long, the PCR program was the following:

PCR settings

PCR setting
Step	Temperature	Time	Go to
1	98°C	30 sec
2	98°C	10 sec
3	72°C	3 sec	step #2, 30 times
4	72°C	10 min
5	4°C	pause

Since R0010 is very short, the PCR products were run on a 1.5% agarose gel using transparent loading dye.

Gel

Loading scheme
100bp ladder
AHF1
AHF2
AHF3
AGC1
AGC2
empty
1kb ladder

$\"Gel\"$

As shown in the gel, a band at nearly 200bp is present in each lane. So, the PCRs were successful!

(4) linear pSB1C3 amplification

I didn't know that linear pSB1C3 is \"impossible\" to amplify, and that the only way to get it is to linearize the circular one. So I tried its amplification and failed.

Gel

Loading scheme
1kb ladder	G3A	G3B	G3C

$\"Gel\"$

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

@@ Line 1: / Line 1: @@
-{
+{"date" : "2013-07-02","author" : "gabriele","title" : "Another busy day","content" : "<html><h3>SAM synthetase ligation in (linear) pSB1C3</h3>First of all, early in the morning I added 1&micro;l of DpnI to the SAMsynthetase#G2 O/N digestion and 1&micro;l of SAP to the linear_pSB1C3#G2 digestion and then incubated them at 37&deg;C for 1.5 hours (afterward I inhibited the enzyme with 20 minutes at 80&deg;C).<br/><br/>Then, I purified and quantified the two digestion samples:<center><table class=\"tn-sp-table\"><tr><th>Sample</th><th>Quantity</th></tr><tr><td>G1 <i>SAMsynthetase</i></td><td>18.8ng/&micro;l</td></tr><tr><td>G2 <i>linear pSB1C3</i></td><td>6.5ng/&micro;l</td></tr></table></center><br/>After that, I performed the ligation (the plasmid had a too low concentration).<center><table class=\"tn-sp-table\"><tr><td style=\"border:none\"></td><th>Ctrl</th><th>0.5:1</th><th>0.5:2</th></tr><tr><td>Buffer</td><td colspan=\"3\">3&micro;l</td></tr><tr><td>Plasmid</td><td colspan=\"3\">14.3&micro;l</td></tr><tr><td>Insert</td><td>0</td><td>6&micro;l</td><td>12&micro;l</td></tr><tr><td>Ligase</td><td colspan=\"3\">1&micro;l</td></tr><tr><td>Water</td><td>11.7&micro;l</td><td>5.7&micro;l</td><td>0&micro;l</td></tr></table></center><br/>And, \"finally\", I transformed the ligations in NEB10&beta; cells.<br/><br/><hr><h3>(2) SAMsynthetase amplification</h3>I amplified SAMsynthetase from the E2 sample (that was purified <a href=\"https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook#tn-post-2013-07-01-Gabriele\">yesterday</a>) following the <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#SAM-extraction-genome-coli\">usual protocol</a>. The PCR was performed in triplicates.<br/><br/>The PCR products were run on a 1% agarose gel.</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Gel|<html><center><table class=\"tn-sp-table\"><tr><th colspan=\"5\">Loading scheme</th></tr><tr><td>E1A</td><td>E1B</td><td>E1C</td><td><i>empty</i></td><td>1kb ladder</td></tr></table><img src=\"https://static.igem.org/mediawiki/2013/6/6e/Tn-20130702-GG_SAMsynthetase_linearpSB1C3.jpg\" alt=\"Gel\" width=\"450px\"></center></html>}}<html><br/>As shown in the gel, a band at nearly 1200bp is present, confirming the success of the PCR.<br/><br/><hr><h3>(3) R0010 amplification</h3>I also amplified R0010 from <b>A</b> sample (R0010, 243.7ng/&micro;l) from <a href=\"https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook#tn-post-2013-06-27-gabriele-emil\">27/06</a>. Being the first time amplifying this sequence, I performed (in triplicates or duplicates) a Phusion PCR using as primers the prefix Fw (Tm = 86&deg;C) and the suffix Rv (Tm = 90&deg;C).<br/></html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|PCR Mixes|<html><center><table class=\"tn-sp-table\"><tr><td style=\"border:none;\"></td><th>Mix HF (x3)</th><th>Mix GC (x2)</th></tr><tr><td>Phusion GC buffer</td><td>0</td><td>10&micro;l</td></tr><tr><td>Phusion HF buffer</td><td>10&micro;l</td><td>0</td></tr><tr><td>dNTPs</td><td colspan=\"2\">1&micro;l</td></tr><tr><td>primer Fw</td><td colspan=\"2\">2.5&micro;l</td></tr><tr><td>primer Rv</td><td colspan=\"2\">2.5&micro;l</td></tr><tr><td>template (50ng/&micro;l)</td><td colspan=\"2\">0.5&micro;l</td></tr><tr><td>Phusion pol</td><td colspan=\"2\">0.5&micro;l</td></tr><tr><td>Water</td><td colspan=\"2\">33&micro;l</td></tr></table></center></html>}}<html><br/>Given that R0010 is 200bp long, the PCR program was the following:</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|PCR settings|<html><center><table class=\"tn-sp-table\"><tr><th colspan=\"4\">PCR setting</th></tr><tr><th>Step</th><th>Temperature</th><th>Time</th><th>Go to</th></tr><tr><td>1</td><td>98&deg;C</td><td>30 sec</td><td></td></tr><tr><td>2</td><td>98&deg;C</td><td>10 sec</td><td></td></tr><tr><td>3</td><td>72&deg;C</td><td>3 sec</td><td>step #2, 30 times</td></tr><tr><td>4</td><td>72&deg;C</td><td>10 min</td><td></td></tr><tr><td>5</td><td>4&deg;C</td><td>pause</td><td></td></tr></table></center></html>}}<html><br/>Since R0010 is very short, the PCR products were run on a 1.5% agarose gel using transparent loading dye.</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Gel|<html><center><table class=\"tn-sp-table\"><tr><th>Loading scheme</th></tr><tr><td>100bp ladder</td></tr><tr><td>AHF1</td></tr><tr><td>AHF2</td></tr><tr><td>AHF3</td></tr><tr><td>AGC1</td></tr><tr><td>AGC2</td></tr><tr><td><i>empty</i></td><tr><td>1kb ladder</td></tr></tr></table><img src=\"https://static.igem.org/mediawiki/2013/3/36/Tn-20130702-GG_R0010.jpg\" alt=\"Gel\" width=\"450px\"></center></html>}}<html><br/>As shown in the gel, a band at nearly 200bp is present in each lane. So, the PCRs were successful!<br/><br/><hr><h3>(4) linear pSB1C3 amplification</h3>I didn't know that linear pSB1C3 is \"<i>impossible</i>\" to amplify, and that the only way to get it is to linearize the circular one. So I tried its amplification and failed.</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>G3A</td><td>G3B</td><td>G3C</td></tr></table><img src=\"https://static.igem.org/mediawiki/2013/6/6e/Tn-20130702-GG_SAMsynthetase_linearpSB1C3.jpg\" alt=\"Gel\" width=\"450px\"></center></html>}}<html></html>","tags" : "SAMsynthetase-Plac-pSB1C3"}
-	"date" : "2013-07-19",
-	"author" : "emil-viola",
-	"title" : "The incompetent Bacillus subtilis saga OD(io)",
-	"content" : "<html>We have tied to grow B.subtilis in the minimal medium (Cambridge protocol and Mansy's friend's protocol) with no evidence of growth, in the previous days we have observed that B. subtilis grow in LB so we have tried to transform B. subtilis in  LB following the LMU munich protocol modified.We transformed 2 groups of cells with the construct K823026+GFP but we mistaken the right Kanamicin concentration of the stock solution(10mg/ml)so no the following day ther were no colonies on the plates.</html>",
-	"tags" : "Bacillus subtilis"
-}