Difference between revisions of "Team:Paris Saclay/Notebook/September/12"
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= Monday 12<sup>th</sup> September= | = Monday 12<sup>th</sup> September= | ||
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===Visualization=== | ===Visualization=== | ||
| − | ==== Glycerol stocks of clones 4, 9, 13 and 16 containing FRB - GFP11 in pSB1C3 ==== | + | ==== Glycerol stocks of clones 4, 9, 13 and 16 containing FRB - GFP11 in pSB1C3 (pPS16_019) ==== |
| − | "By Maxence" | + | "By Maxence & Mahnaz" |
The glycerol stock of the bacteria with the following plasmids were made. | The glycerol stock of the bacteria with the following plasmids were made. | ||
| − | * | + | *pPS16_019 (FRB - GFP11) clone 4 |
| − | * | + | *pPS16_019 (FRB - GFP11) clone 9 |
| − | * | + | *pPS16_019 (FRB - GFP11) clone 13 |
| − | * | + | *pPS16_019 (FRB - GFP11) clone 16 |
500µL of liquid culture and 250 µL of glycerol 60% were mixed and put at -20°C. | 500µL of liquid culture and 250 µL of glycerol 60% were mixed and put at -20°C. | ||
| − | + | ====Plasmids extraction of clones 4, 9, 13 and 16 containing FRB - GFP11 in pSB1C3 (pPS16_019)==== | |
| − | + | "By Maxence & Mahnaz" | |
| − | ====Plasmids extraction of clones 4, 9, 13 and 16 containing FRB - GFP11 in pSB1C3==== | + | |
| − | "By Maxence" | + | |
The following plasmids were extracted using the [[Team:Paris_Saclay/Experiments#InvitrogenPlasmidExtraction|"Charge Switch-Pro Plasmid Miniprep"]] kit: | The following plasmids were extracted using the [[Team:Paris_Saclay/Experiments#InvitrogenPlasmidExtraction|"Charge Switch-Pro Plasmid Miniprep"]] kit: | ||
| − | * | + | *pPS16_019 (FRB - GFP11) clone 4 |
| − | * | + | *pPS16_019 (FRB - GFP11) clone 9 |
| − | * | + | *pPS16_019 (FRB - GFP11) clone 13 |
| − | * | + | *pPS16_019 (FRB - GFP11) clone 16 |
====Samples preparation for sequencing==== | ====Samples preparation for sequencing==== | ||
| − | ''By Maxence'' | + | ''By Maxence & Mahnaz'' |
| − | 20 µL of plasmids pSB1C3 FRB - GFP 11 (clones 4, 9, 13 and 16) were sent to be sequenced. 20 µL of the primers iPS83 (5µM) and iPS84 (5µM) were sent for sequencing. | + | 20 µL of plasmids pSB1C3 FRB - GFP 11 (pPS16_019) (clones 4, 9, 13 and 16) were sent to be sequenced. 20 µL of the primers iPS83 (5µM) and iPS84 (5µM) were sent for sequencing. |
====NanoDrop Measurements==== | ====NanoDrop Measurements==== | ||
| − | ''By Maxence'' | + | ''By Maxence & Mahnaz'' |
{| class="wikitable" | {| class="wikitable" | ||
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====Gibson of cleaned up PCR products GFP 1.9 from 9th September x pSB1C3 treated by DpnI from 22nd August==== | ====Gibson of cleaned up PCR products GFP 1.9 from 9th September x pSB1C3 treated by DpnI from 22nd August==== | ||
| − | ''By Maxence'' | + | ''By Maxence & Mahnaz'' |
Gibson was performed with cleaned up PCR products GFP 1.9 (insert) (obtained the 9th September) x pSB1C3 treated by DpnI (plasmid) (obtained the 22nd August) with the following protocol: | Gibson was performed with cleaned up PCR products GFP 1.9 (insert) (obtained the 9th September) x pSB1C3 treated by DpnI (plasmid) (obtained the 22nd August) with the following protocol: | ||
| Line 82: | Line 82: | ||
Two controls were done: one without insert and one without buffer (water was added in order to have a total of 20 µL). The PCR was performed as follow : 1 hour at 50°C. | Two controls were done: one without insert and one without buffer (water was added in order to have a total of 20 µL). The PCR was performed as follow : 1 hour at 50°C. | ||
| − | ====Transformation of DH5a cells with GFP 1.9 in pSB1C3 obtained by Gibson==== | + | ====Transformation of DH5a cells with GFP 1.9 in pSB1C3 (pPS16_020) obtained by Gibson==== |
| − | ''By Maxence'' | + | ''By Maxence & Mahnaz'' |
Dh5a cells were transformed with pSB1C3 containing GFP 1.9, or controls (no buffer mix and plasmid alone) using the usual [[Team:Paris_Saclay/Experiments#heat-shocktransformation|protocol]]. | Dh5a cells were transformed with pSB1C3 containing GFP 1.9, or controls (no buffer mix and plasmid alone) using the usual [[Team:Paris_Saclay/Experiments#heat-shocktransformation|protocol]]. | ||
====gblock 1.1 - 1.2 and gblock 2.1 - 2.2 Ligation==== | ====gblock 1.1 - 1.2 and gblock 2.1 - 2.2 Ligation==== | ||
| − | ''By Maxence'' | + | ''By Maxence & Mahnaz'' |
| − | gBlocks | + | gBlocks pPS16_001 and pPS16_002 were ligated together as following : |
| − | * 8 µL of | + | * 8 µL of pPS16_001 PCR product from 9th September |
| − | * 8 µL of | + | * 8 µL of pPS16_002 PCR product from 9th September |
* 2 µL of Buffer T4 10X | * 2 µL of Buffer T4 10X | ||
* 2 µL of ligase T4 enzyme | * 2 µL of ligase T4 enzyme | ||
| Line 99: | Line 99: | ||
gBlocks pPS16_003 and pPS16_004 were ligated together as following : | gBlocks pPS16_003 and pPS16_004 were ligated together as following : | ||
| − | * 8 µL of | + | * 8 µL of pPS16_003 PCR product from 9th September |
* 8 µL of pPS16_004 PCR product from 9th September | * 8 µL of pPS16_004 PCR product from 9th September | ||
* 2 µL of Buffer T4 10X | * 2 µL of Buffer T4 10X | ||
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The ligation product was put at rooming temperature for 2 hours. | The ligation product was put at rooming temperature for 2 hours. | ||
| − | |||
| − | |||
====Clean-up of Ligation products ==== | ====Clean-up of Ligation products ==== | ||
| − | ''By Maxence'' | + | ''By Maxence & Mahnaz'' |
Ligation products obtained were cleaned up by using the NucleoSpin Gel and PCR Clean-up kit [[Team:Paris_Saclay/Experiments#Purification|protocol]]. | Ligation products obtained were cleaned up by using the NucleoSpin Gel and PCR Clean-up kit [[Team:Paris_Saclay/Experiments#Purification|protocol]]. | ||
====NanoDrop Measurements==== | ====NanoDrop Measurements==== | ||
| − | ''By Maxence'' | + | ''By Maxence & Mahnaz'' |
{| class="wikitable" | {| class="wikitable" | ||
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====PCR of cleaned up Ligation products (fragment 1 and fragment 2) with 3% DMSO ==== | ====PCR of cleaned up Ligation products (fragment 1 and fragment 2) with 3% DMSO ==== | ||
| − | ''By Maxence'' | + | ''By Maxence & Mahnaz'' |
As the annealing temperature (Tm) seems too high to obtain good results for fragments 1 and 2 amplification, DMSO was used in order to reduce the annealing temperature during the PCR. For that purpose, PCR was performed with the following protocol. | As the annealing temperature (Tm) seems too high to obtain good results for fragments 1 and 2 amplification, DMSO was used in order to reduce the annealing temperature during the PCR. For that purpose, PCR was performed with the following protocol. | ||
| Line 175: | Line 173: | ||
|} | |} | ||
| − | + | [[Team:Paris_Saclay/Experiments#primers|Primers]] used were: | |
{| class="wikitable" | {| class="wikitable" | ||
| Line 189: | Line 187: | ||
====Gel of PCR products==== | ====Gel of PCR products==== | ||
| − | ''By Maxence'' | + | ''By Maxence & Mahnaz'' |
After amplification, 3 µL of each cleaned up PCR products and 5 µL of DNA ladder were placed in wells and migrated at 100V during 30 min. | After amplification, 3 µL of each cleaned up PCR products and 5 µL of DNA ladder were placed in wells and migrated at 100V during 30 min. | ||
| Line 208: | Line 206: | ||
|} | |} | ||
| − | + | [[File:T--Paris Saclay--Gel119.png|400px|thumb|center|Result of the migration]] | |
A large smearing in agarose gel of PCR products was obtained. As this result was not usable, a new PCR was run by diluting the Ligations products : 1/100, 1/10,000 and 1/1,000,000. | A large smearing in agarose gel of PCR products was obtained. As this result was not usable, a new PCR was run by diluting the Ligations products : 1/100, 1/10,000 and 1/1,000,000. | ||
| − | + | [[File:T--Paris Saclay--Gel1110.png|400px|thumb|center|Result of the migration]] | |
A large smearing in agarose gel of PCR products was also obtained, but the smears were seemed to decrease with the dilution rate. | A large smearing in agarose gel of PCR products was also obtained, but the smears were seemed to decrease with the dilution rate. | ||
| + | {{Team:Paris_Saclay/notebook_footer}} | ||
Latest revision as of 17:04, 9 October 2016
