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Team:UNITN-Trento/Notebook/Labposts/07/01
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| - | { | + | {"date" : "2013-07-01","author" : "emil","title" : " Re-screening, amplification and digestion(together with the two B. subtilis backbones) of the GFPs E0840 and E0240 ","content" : "<html>I screened the three other sample of GFP(0840/2,0840/4,0240/1,0240/3) like in the<a href=\"https://2013.igem.org/Team:UNITN-Trento/Notebook#tn-post-2013-06-29-emil>\"> previous post </a> with the following results:(the gel was run together with Girelli's sample, we are interested in the first 4 sample)</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Gel order|<html><center><table class=\"tn-sp-table\"><tr><th>Sample</th><th>Well</th></tr><tr><td>BBa_E0840/2</td><td>2</td></tr><tr><td>BBa_E0840/4</td><td>3</td></tr><tr><td>BBa_E0240/1</td><td>4</td></tr><tr><td>BBa_E0240/3</td><td>5</td></tr><tr><td>Ladder 1kb Fermentas</td><td>1</td></tr></table></center></html>}}{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Gel image|<html> <img src=\"https://static.igem.org/mediawiki/2013/e/ef/Tn-20130701-GGET_GFP_PSB1A2R0010SAMsynth.jpg\" /></html>}}<html>We can see 2 bands for each sample: the upper is the plasmid(pSB1A2 2079 bp), the lower is presumably the GFP(BBa_E0240 826 bp,BBa_E0840 828 bp)(prefix and suffix escluded)at 900 bp.Afterwards i have amplified 0.5 6micro;l of one of the 4 sample(E0840/4 475.9 ng/micro;l) following the <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#Phusion-PCR\">PCR protocol</a> and exploiting the universal primers(prefix Forward,Suffix Reverse) with the following resuts(as usual I did a gel to verify the pcr):</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Gel order|<html><center><table class=\"tn-sp-table\"><tr><th>Sample</th><th>Well</th></tr><tr><td>BBa_E0840/4 a</td><td>2</td></tr><tr><td>BBa_E0840/4 b</td><td>3</td></tr><tr><td>BBa_E0840/4 c</td><td>4</td></tr><tr><td>Ladder 1kb Fermentas</td><td>1</td></tr></table></center></html>}}{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Gel image|<html> <img src=\"https://static.igem.org/mediawiki/2013/8/81/Tn-20130701-GGET_GFPpcr_SAMampli.jpg\" width=\"450\" /></html>}}<html> As we can see the pcr completed succesfully, there are the right bands at 900-1000 bp.Then I purified the pcr following the <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#Promega-PCR-Gel\"> purification protocol </a>. After that I have quantified the product with the following results:</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Quantification|<html><center><table class=\"tn-sp-table\"><tr><th>Sample</th><th>Quantification</th></tr><tr><td>BBa_E0840/4 a</td><td>30.5ng/µl</td></tr><tr><td>BBa_E0840/4 b</td><td> 44ng/µl</td></tr><tr><td>BBa_E0840/4 c</td><td>42.6ng/µl</td></tr></table></center></html>}}<html>Finally I have digested o/n all the 48.5µl of b sample and of 2 sample of the backbone(K823026 359.7ng/µl,K823024 334.4ng/µl) with EcoR1-HF and Pst1 following the <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#Digestion\"> digestion protocol</a>. </html>","tags" : "K832024-K823026-E0840-E0240"} |
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Latest revision as of 10:30, 3 October 2013
{"date" : "2013-07-01","author" : "emil","title" : " Re-screening, amplification and digestion(together with the two B. subtilis backbones) of the GFPs E0840 and E0240 ","content" : "I screened the three other sample of GFP(0840/2,0840/4,0240/1,0240/3) like in the\"> previous post with the following results:(the gel was run together with Girelli's sample, we are interested in the first 4 sample)
Gel order
We can see 2 bands for each sample: the upper is the plasmid(pSB1A2 2079 bp), the lower is presumably the GFP(BBa_E0240 826 bp,BBa_E0840 828 bp)(prefix and suffix escluded)at 900 bp.Afterwards i have amplified 0.5 6micro;l of one of the 4 sample(E0840/4 475.9 ng/micro;l) following the PCR protocol and exploiting the universal primers(prefix Forward,Suffix Reverse) with the following resuts(as usual I did a gel to verify the pcr):| Sample | Well |
|---|---|
| BBa_E0840/2 | 2 |
| BBa_E0840/4 | 3 |
| BBa_E0240/1 | 4 |
| BBa_E0240/3 | 5 |
| Ladder 1kb Fermentas | 1 |
![](\"https://static.igem.org/mediawiki/2013/e/ef/Tn-20130701-GGET_GFP_PSB1A2R0010SAMsynth.jpg\")
Gel order
As we can see the pcr completed succesfully, there are the right bands at 900-1000 bp.Then I purified the pcr following the purification protocol . After that I have quantified the product with the following results:| Sample | Well |
|---|---|
| BBa_E0840/4 a | 2 |
| BBa_E0840/4 b | 3 |
| BBa_E0840/4 c | 4 |
| Ladder 1kb Fermentas | 1 |
![](\"https://static.igem.org/mediawiki/2013/8/81/Tn-20130701-GGET_GFPpcr_SAMampli.jpg\")
Quantification
Finally I have digested o/n all the 48.5µl of b sample and of 2 sample of the backbone(K823026 359.7ng/µl,K823024 334.4ng/µl) with EcoR1-HF and Pst1 following the digestion protocol. ","tags" : "K832024-K823026-E0840-E0240"}
| Sample | Quantification |
|---|---|
| BBa_E0840/4 a | 30.5ng/µl |
| BBa_E0840/4 b | 44ng/µl |
| BBa_E0840/4 c | 42.6ng/µl |
