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Team:UNITN-Trento/Notebook/Labposts/07/02
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| - | { | + | {"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/µl</td></tr><tr><td>E2</td><td>60ng/µl</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>0.63µl</td><td>0.83µl</td></tr><tr><td>dNTPs</td><td colspan=\"2\">0.5µl</td></tr><tr><td>primer Fw</td><td colspan=\"2\">1µl</td></tr><tr><td>primer Rv</td><td colspan=\"2\">1µl</td></tr><tr><td>buffer RBC</td><td colspan=\"2\">5µl</td></tr><tr><td>Phusion pol</td><td colspan=\"2\">0.3µl</td></tr><tr><td>RBC pol</td><td colspan=\"2\">0.25µl</td></tr><tr><td>water</td><td>41.32µl</td><td>41.12µl</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/µl</td></tr><tr><td>E2</td><td>SAM synthetase</td><td>31.5ng/µl</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/µl</td></tr><tr><td>B</td><td>252.1ng/µl</td></tr><tr><td>C</td><td>345.6ng/µl</td></tr><tr><td>D</td><td>295.3ng/µl</td></tr><tr><td>E</td><td>315.6ng/µl</td></tr><tr><td>F</td><td>226.2ng/µl</td></tr><tr><td rowspan=\"3\">1:3</td><td>A</td><td>345.7ng/µl</td></tr><tr><td>B</td><td>147.0ng/µl</td></tr><tr><td>C</td><td>268.0ng/µl</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/µl</td></tr><tr><td>G3</td><td>linear pSB1C3</td><td>63.3ng/µl</td></tr><tr><td>G2A</td><td>linear pSB1C3</td><td>44.0ng/µl</td></tr><tr><td>G3A</td><td>linear pSB1C3</td><td>44.8ng/µl</td></tr><tr><td>G4A</td><td>linear pSB1C3</td><td>41.9ng/µl</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 (3µg)</td><td>48.5µl</td><td>40µl</td></tr><tr><td>XbaI</td><td>2.5µl</td><td>1.5µl</td></tr><tr><td>PstI</td><td>2.5µl</td><td>1.5µl</td></tr><tr><td>NEBuffer 2</td><td>10µl</td><td>5µl</td></tr><tr><td>water</td><td>26.5µl</td><td>0</td></tr></table></center></html>}}<html></html>","tags" : "SAMsynthetase-Plac-pSB1A2-pSB1C3"} |
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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
![\"Gel\"](\"https://static.igem.org/mediawiki/2013/8/81/Tn-20130701-GGET_GFPpcr_SAMampli.jpg\")
| Loading scheme | ||
|---|---|---|
| 1kb ladder | G1 | E2 |
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
![\"gel\"](\"https://static.igem.org/mediawiki/2013/e/ef/Tn-20130701-GGET_GFP_PSB1A2R0010SAMsynth.jpg\")
| 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 |
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
","tags" : "SAMsynthetase-Plac-pSB1A2-pSB1C3"}
| 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 |
