Line 44:
Line 44: The mix were incubated for 1 hour at 37°C. Then, 20 µL of digestion products and 15 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.
The mix were incubated for 1 hour at 37°C. Then, 20 µL of digestion products and 15 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.
− GEL SYLVIE 1
+ [[File:T--Paris Saclay--Gel111111112.png|400px|thumb|center|Result of the migration]]
The digestions were good for pPS16_019, pPS16_018 and BbaB0015, but we did not obtained the expecting band size for pPS16_009.
The digestions were good for pPS16_019, pPS16_018 and BbaB0015, but we did not obtained the expecting band size for pPS16_009.
Revision as of 14:47, 18 October 2016
Monday 10th October Visualization Plasmids extraction of FRB - GFP 11 in pSB1C3 (pPS16_019) clone 4, FKBP - GFP 10 in pSB1C3 (pPS16_018) clone 6, GFP 1.9 in pUC19 (pPS16_009) clone 1 and BbaB0015 By Sylvie
The OD were measured for each overnight cultures:
Sample
OD (1 ml)
FRB - GFP 11 in pSB1C3 (pPS16_019)
3
FKBP - GFP 10 in pSB1C3 (pPS16_018)
3.9
GFP 1.9 in pUC19 (pPS16_009)
4.2
BbaB0015
5
"standard Plasmid Miniprep" .
By Maxence & Caroline
Extracted pPS16_019, pPS16_018, pPS16_009 and BbaB0015 from the 10th October were digested by restriction enzymes EcoRI in order to verify the concentration as following:
2 µL of plasmid
2 µL of buffer FD
2 µL of restriction enzyme EcoRI
14 µL of water The mix were incubated for 1 hour at 37°C. Then, 20 µL of digestion products and 15 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.
The digestions were good for pPS16_019, pPS16_018 and BbaB0015, but we did not obtained the expecting band size for pPS16_009.
BioBrick K2039000 and K2039001 characterization Protein extraction of FRB - GFP 11 in pSB1C3 (pPS16_019) clone 4, FKBP - GFP 10 in pSB1C3 (pPS16_018) clone 6, GFP 1.9 in pUC19 (pPS16_009) clone 1 and PhB1040 strain 594 By Maxence
In order to perform a Western Blot, the proteins were extracted from clones (pPS16_018 clone 6, pPS16_019 clone 4, pPS16_009 clone 1 and PhB1040 strain 594) put on liquide culture the 7th October. For that purpose, the protein extraction protocol was used:
Sample
OD (1 ml)
Volume of lysis buffer
FRB - GFP 11 in pSB1C3 (pPS16_019)
5.1
127.5 μl
FKBP - GFP 10 in pSB1C3 (pPS16_018)
5.2
130 μl
GFP 1.9 in pUC19 (pPS16_009)
5.3
132.5 μl
PhB1040 strain 594
2.8
70 μl
Protein electrophoresis of FRB - GFP 11 in pSB1C3 (pPS16_019) clone 4, FKBP - GFP 10 in pSB1C3 (pPS16_018) clone 6, GFP 1.9 in pUC19 (pPS16_009) clone 1 and PhB1040 strain 594 By Maxence
A protein electrophoresis of the extracted protein was done using the protein electrophoresis protocol . For that purpose, the gel cassette Bolt 4-12% Bis-Tris Plus 10W was used and the power was set for 1 hour at 65V.
Transfert of protein extracted from FRB - GFP 11 in pSB1C3 (pPS16_019) clone 4, FKBP - GFP 10 in pSB1C3 (pPS16_018) clone 6, GFP 1.9 in pUC19 (pPS16_009) clone 1 and PhB1040 strain 594 By Maxence
The proteins on the electrophoresis gel were transfered on blotting membrane using the protein transfert protocol .
