(Created page with "{{Team:Paris_Saclay/notebook_header}} = Tuesday 4<sup>th</sup> October= ==Lab work== ===Visualization=== ====Colony PCR of clones containing FRB - GFP 11 - FKBP - GFP 10 in...") |
(→PCR of extracted GFP 1.9 in pSB1C3 (pPS16_009)) |
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= Tuesday 4<sup>th</sup> October= | = Tuesday 4<sup>th</sup> October= | ||
− | + | ||
===Visualization=== | ===Visualization=== | ||
− | ==== | + | ====Culture of FRB - GFP 11 in pSB1C3 (pPS16_019) clone 4, FKBP - GFP 10 in pSB1C3 (pPS16_018) clone 6 and GFP 1.9 in pUC19 (pPS16_009) clone 1==== |
''By Maxence'' | ''By Maxence'' | ||
− | + | As no clones containing FRB - GFP 11 - FKBP - GFP 10 in pSB1C3 (pPS16_021) were obtained and as the concentrations of plasmids extracted the 2nd October were too low, the following clones were put on 2 x 10 mL liquid cultures (LB + 30 µg/mL Cm) grown at 37°C overnight: | |
− | + | *pPS16_018 (FKBP - GFP 10) clone 6 | |
− | * | + | *pPS16_019 (FRB - GFP 11) clone 4 |
− | + | ||
− | * | + | |
− | + | ||
− | PCR was | + | ====PCR of extracted GFP 1.9 in pUC19 (pPS16_009)==== |
+ | ''By Maxence'' | ||
+ | |||
+ | As no good clones were obtained for GFP 1.9 in pSB1C3 (pPS16_020), a new PCR was run using extracted plasmids containing GFP 1.9 in pUC19 (pPS16_009) extracted the 2nd October. For each amplification, two matrix were used as two liquid cultures were set the 1st October. | ||
+ | |||
+ | For each 50μl of reaction, mix the following reagents : | ||
+ | * 2 µL of matrix | ||
+ | * 1 µL of dNTPs (10mM) | ||
+ | * 2.5 µL of each primer mix (10µM) | ||
+ | * 10 µL of buffer (5X) | ||
+ | * 0,5 µL of Phusion polymerase | ||
+ | * 30 µL of nuclease free water | ||
+ | * 1.5 µL of DMSO | ||
+ | |||
+ | Mix gently and aliquot 50 μl of the mix into the PCR tubes on ice. | ||
+ | Perform PCR as follow: | ||
{| class="wikitable" | {| class="wikitable" | ||
|- | |- | ||
Line 24: | Line 36: | ||
|- | |- | ||
|Initial denaturation | |Initial denaturation | ||
− | | | + | |98°C |
− | | | + | |30sec |
|- | |- | ||
|rowspan="3"|30 cycles | |rowspan="3"|30 cycles | ||
− | | | + | |98°C |
− | | | + | |10sec |
|- | |- | ||
− | | | + | |60°C |
− | | | + | |30sec |
|- | |- | ||
|72°C | |72°C | ||
− | | | + | |30 sec |
|- | |- | ||
|Final Extension | |Final Extension | ||
|72°C | |72°C | ||
− | | | + | |2min |
|- | |- | ||
|Hold | |Hold | ||
Line 51: | Line 63: | ||
|- | |- | ||
!Matrix | !Matrix | ||
− | ! | + | !GFP 1.9 in pUC19 (pPS16_009) extracted the 2nd October |
|- | |- | ||
|Primers | |Primers | ||
− | | | + | |iPS84 and iPS140 |
|- | |- | ||
|} | |} | ||
− | + | 5 µL of PCR products and 20 µL of DNA ladder were placed in wells and migrated at 100V during 30 min, in odrder to verify the DNA quantity. | |
PCR products expected were : | PCR products expected were : | ||
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!Expected band size (bp) | !Expected band size (bp) | ||
|- | |- | ||
− | | | + | |GFP 1.9 |
− | | | + | |862 |
+ | |- | ||
|} | |} | ||
− | + | [[File:T--Paris Saclay--Gel11111119.png|400px|thumb|center|Result of the migration]] | |
+ | |||
+ | ====Cloning of GFP 1.9 PCR product in pSB1C3 by digestion-ligation==== | ||
+ | ''By Maxence'' | ||
+ | |||
+ | For that purpose, GFP 1.9 PCR product from today was cut by restriction enzymes PstI & XbaI as following: | ||
+ | |||
+ | For the most concentrated one (GFP 1.9 - 1): | ||
+ | |||
+ | * 10 µL of GFP 1.9 PCR product | ||
+ | * 4 µL of buffer FD | ||
+ | * 1 µL of restriction enzyme PstI | ||
+ | * 1 µL of restriction enzyme XbaI | ||
+ | * 24 µL of water | ||
+ | |||
+ | For the less concentrated one (GFP 1.9 - 2): | ||
+ | |||
+ | * 20 µL of GFP 1.9 PCR product | ||
+ | * 4 µL of buffer FD | ||
+ | * 1 µL of restriction enzyme PstI | ||
+ | * 1 µL of restriction enzyme XbaI | ||
+ | * 14 µL of water | ||
+ | |||
+ | Furthermore, BbaB0015 plasmid (in order to have pSB1C3) was cut by restriction enzymes PstI & XbaI and was treated by the alkaline phosphatase FastAP (dephosphorylation of cloning vector DNA to prevent recircularization during ligation) as following: | ||
+ | |||
+ | * 20 µL of BbaB0015 plasmid | ||
+ | * 4 µL of buffer FD | ||
+ | * 1 µL of restriction enzyme EcoRI | ||
+ | * 1 µL of restriction enzyme XbaI | ||
+ | * 1 µL of alkaline phosphatase FastAP | ||
+ | * 13 µL of water | ||
+ | |||
+ | The mix were incubated for 1 hour at 37°C. Then, 5 µL of each digestion products and 20 µL of DNA ladder were placed in wells and migrated at 100V during 30 min. | ||
+ | |||
+ | Migration products expected were : | ||
+ | |||
+ | {| class="wikitable" | ||
+ | |- | ||
+ | !Migration products | ||
+ | !Expected band size (bp) | ||
+ | |- | ||
+ | |Template digested (GFP 1.9 PCR product treated by PstI & XbaI) | ||
+ | |900 | ||
+ | |- | ||
+ | |Vector digested (BbaB0015 treated by PstI & XbaI) | ||
+ | |2000 | ||
+ | |- | ||
+ | |} | ||
+ | |||
+ | [[File:T--Paris Saclay--Gel111111110.png|400px|thumb|center|Result of the migration]] | ||
+ | |||
+ | The digestion products were cleaned up by using the NucleoSpin Gel and PCR Clean-up kit [[Team:Paris_Saclay/Experiments#Purification|protocol]]. After cleaned-up, sample from extract 1 and sample from extract 2 were pooled together. Then 2 µL of each cleaned-up products and 20 µL of DNA ladder were placed in wells and migrated at 100V during 30 min. | ||
+ | |||
+ | [[File:T--Paris Saclay--Gel111111111.png|400px|thumb|center|Result of the migration]] | ||
+ | |||
+ | Once template and vector were cut and purified, DNA ligase was used to join the sticky ends of the template and vector together: | ||
+ | |||
+ | * 9 µL of template (GFP 1.9 PCR product treated by PstI & XbaI) | ||
+ | * 3 µL of vector (BbaB0015 treated by PstI & XbaI) | ||
+ | * 2 µL of Buffer T4 10X | ||
+ | * 1 µL of ligase T4 enzyme | ||
+ | * 5 µL of water | ||
+ | |||
+ | A control was done without template. The mix were incubated for 1 hour at rooming temperature. Then the ligase was desactivated at 75°C for 10 minutes. | ||
+ | |||
+ | ====Transformation of DH5a cells with and GFP 1.9 in pSB1C3 (pPS16_020) obtained by Digestion-Ligation==== | ||
+ | ''By Maxence'' | ||
+ | |||
+ | Dh5a cells were transformed with pSB1C3 containing GFP 1.9 (pPS16_020) or controls (digested plasmid) using the usual [[Team:Paris_Saclay/Experiments#heat-shocktransformation|protocol]]. | ||
{{Team:Paris_Saclay/notebook_footer}} | {{Team:Paris_Saclay/notebook_footer}} |
Latest revision as of 12:09, 18 October 2016