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Team:UNITN-Trento/Notebook/Labposts/06/28
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{ | { | ||
| - | "date" : "2013-06- | + | "date" : "2013-06-14", |
| - | "author" : " | + | "author" : "gabriele-emil", |
| - | "title" : " | + | "title" : "What happend to SAMsynthetase ligation?", |
| - | "content" : " <html> | + | "content" : "<html>Got the inocula from yesterday:<ul><li>4 out of 5 inocula of SAMsynthetase+pSB1C3[no_RFP] were good, one (3) didn't grow.</li><li>4 out of 5 inocula of SAMsynthetase+pSB1C3[RFP] were good, one (5R) was red!</li></ul></html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|SAMsynthetase+pSB1C3[no_RFP] Inocula|<html><center><img src=\"https://static.igem.org/mediawiki/2013/9/91/Tn-20130614-SAM_inocula.JPG\" width=\"400px\" /></center></html>}}<html></html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|SAMsynthetase+pSB1C3[RFP] Inocula|<html><center><img src=\"https://static.igem.org/mediawiki/2013/2/2e/Tn-20130614-SAM_RFP_inocula.JPG\" width=\"400px\" /></center></html>}}<html></html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|5R (red) Inocula|<html><center><img src=\"https://static.igem.org/mediawiki/2013/4/44/Tn-20130614-SAM_RFP_red_inocula.JPG\" width=\"400px\" /></center></html>}}<html><br/>Then, we miniprepped the samples (except for the red, 5R, one) using the <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#miniprep\">protocol</a>. We chose to miniprep also the inocula that did not grow (3), just as a control. After miniprep, quantification was performed with nanodrop.<br/></html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Quantification results|<html><center><table class=\"tn-sp-table\"><tr><th>Sample</th><th>DNA [ng/µl]</th><th>Sample</th><th>DNA [ng/µl]</th></tr><tr><td>1</td><td>173.7</td><td>1R</td><td>157.0</td></tr><tr><td>2</td><td>188.2</td><td>2R</td><td>97.1</td></tr><tr><td>3</td><td>18.6</td><td>3R</td><td>148.0</td></tr><tr><td>4</td><td>178.7</td><td>4R</td><td>143.7</td></tr><tr><td>5</td><td>103.3</td><td>5R</td><td>-</td></tr></table></center></html>}}<html><br/>After that, we digested an aliquote of 800ng of each DNA sample with EcoRI and PstI using our <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#Digestion\">digestion protocol</a>, putting the digestion mix to incubate for 1.5h at 37°C (static).<br/></html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Digestion mix|<html><center><table class=\"tn-sp-table\"><tr><td style=\"border: none;\"></td><th>1</th><th>2</th><th>4</th><th>5</th><th>1R</th><th>2R</th><th>3R</th><th>4R</th></tr><tr><td>Buffer 10X (Nebuffer4)</td><td colspan=\"8\">2µl</td></tr><tr><td>EcoRI</td><td colspan=\"8\">1µl</td></tr><tr><td>PstI</td><td colspan=\"8\">1µl</td></tr><tr><td>BSA 10X</td><td colspan=\"8\">1µl</td></tr><tr><td>Template</td><td>4.6µl</td><td>4.3µl</td><td>4.3µl</td><td>7.75µl</td><td>5.1µl</td><td>8.2µl</td><td>5.4µl</td><td>5.6µl</td></tr><tr><td>Water (up to 20µl)</td><td>10.4µl</td><td>10.7µl</td><td>10.7µl</td><td>7.25µl</td><td>9.9µl</td><td>6.8µl</td><td>9.6µl</td><td>9.4µl</td></tr><tr><td>Total volume</td><td colspan=\"8\">20µl</td></tr><tr><td>BamHI</td><td colspan=\"8\">1µl</td></tr></table></center></html>}}<html><br/>Then we realized that LacI+RFP and SAMsynthetase have the same base length, and that a gel would not be able to discriminate between them. So, we added 1µl of BamHI to each sample, knowing that this enzyme is able to cut SAMsynthetase (in a band of 100bp and one of 1055bp) to identify the presence of SAM.<br/><br/>So, we prepared a 1.5% agarose gel and performed an electrophoresis for 30 minutes at 120V.</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Gel Image|<html><center><table class=\"tn-sp-table\"><tr><td colspan=\"10\">Loaded samples</td></tr><tr><td>1kb ladder</td><td>#1</td><td>#2</td><td>#4</td><td>#5</td><td>100bp ladder</td><td>#1R</td><td>#2R</td><td>#3R</td><td>#4R</td></tr></table><img src=\"https://static.igem.org/mediawiki/2013/d/d0/Tn-20130614-SAM_gel.JPG\" width=\"400px\" /></center></html>}}<html>From the gel is clear that the samples without RFP (1, 2, 4, 5) do not have any insert. The RFP samples (1R, 2R, 3R, 4R) might contain the SAMsynthetase, but the insert might be LacI+RFP.<br/><br/>To determine whether the insert was LacI+RFP or SAMsynthetase we wanted to perform a <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#RBC-Taq-DNA-pol-PCR\">RBC Taq PCR</a> against SAMsynthetase (SAMsynthetase-Fw and SAMsynthetase-Rv primers), but we set a wrong PCR program... <b>next time verify the PCR program more accurately!!!</b> On Monday we will start again from the PCR... so sad D:<br/></html>", |
| - | "tags" : " | + | "tags" : "SAMsynthetase" |
} | } | ||
Latest revision as of 07:47, 3 October 2013
{ "date" : "2013-06-14", "author" : "gabriele-emil", "title" : "What happend to SAMsynthetase ligation?", "content" : "Got the inocula from yesterday:
- 4 out of 5 inocula of SAMsynthetase+pSB1C3[no_RFP] were good, one (3) didn't grow.
- 4 out of 5 inocula of SAMsynthetase+pSB1C3[RFP] were good, one (5R) was red!
![](\"https://static.igem.org/mediawiki/2013/9/91/Tn-20130614-SAM_inocula.JPG\")
![](\"https://static.igem.org/mediawiki/2013/2/2e/Tn-20130614-SAM_RFP_inocula.JPG\")
![](\"https://static.igem.org/mediawiki/2013/4/44/Tn-20130614-SAM_RFP_red_inocula.JPG\")
Then, we miniprepped the samples (except for the red, 5R, one) using the protocol. We chose to miniprep also the inocula that did not grow (3), just as a control. After miniprep, quantification was performed with nanodrop.
Quantification results
| Sample | DNA [ng/µl] | Sample | DNA [ng/µl] |
|---|---|---|---|
| 1 | 173.7 | 1R | 157.0 |
| 2 | 188.2 | 2R | 97.1 |
| 3 | 18.6 | 3R | 148.0 |
| 4 | 178.7 | 4R | 143.7 |
| 5 | 103.3 | 5R | - |
After that, we digested an aliquote of 800ng of each DNA sample with EcoRI and PstI using our digestion protocol, putting the digestion mix to incubate for 1.5h at 37°C (static).
Digestion mix
| 1 | 2 | 4 | 5 | 1R | 2R | 3R | 4R | |
|---|---|---|---|---|---|---|---|---|
| Buffer 10X (Nebuffer4) | 2µl | |||||||
| EcoRI | 1µl | |||||||
| PstI | 1µl | |||||||
| BSA 10X | 1µl | |||||||
| Template | 4.6µl | 4.3µl | 4.3µl | 7.75µl | 5.1µl | 8.2µl | 5.4µl | 5.6µl |
| Water (up to 20µl) | 10.4µl | 10.7µl | 10.7µl | 7.25µl | 9.9µl | 6.8µl | 9.6µl | 9.4µl |
| Total volume | 20µl | |||||||
| BamHI | 1µl |
Then we realized that LacI+RFP and SAMsynthetase have the same base length, and that a gel would not be able to discriminate between them. So, we added 1µl of BamHI to each sample, knowing that this enzyme is able to cut SAMsynthetase (in a band of 100bp and one of 1055bp) to identify the presence of SAM.
So, we prepared a 1.5% agarose gel and performed an electrophoresis for 30 minutes at 120V.
Gel Image
![](\"https://static.igem.org/mediawiki/2013/d/d0/Tn-20130614-SAM_gel.JPG\")
From the gel is clear that the samples without RFP (1, 2, 4, 5) do not have any insert. The RFP samples (1R, 2R, 3R, 4R) might contain the SAMsynthetase, but the insert might be LacI+RFP.| Loaded samples | |||||||||
| 1kb ladder | #1 | #2 | #4 | #5 | 100bp ladder | #1R | #2R | #3R | #4R |
To determine whether the insert was LacI+RFP or SAMsynthetase we wanted to perform a RBC Taq PCR against SAMsynthetase (SAMsynthetase-Fw and SAMsynthetase-Rv primers), but we set a wrong PCR program... next time verify the PCR program more accurately!!! On Monday we will start again from the PCR... so sad D:
", "tags" : "SAMsynthetase" }
