Bands visible! Lanes 1 and 2 right of the ladder should have a 10506 bp band. Lanes 3 and 4 right of the ladder should have a 11414 bp band (gel description should read EC93 instead of EC90).
PCR cleanup (according to Thermo Scientific protocol), elution in 21 ul.
Concentrations according to Nanodrop: EC93 w/ 001+007: 92 ng/ul; EC93 w/ 001+008: 57 ng/ul
15/05/21
PCR
Primers ICD014 and ICD015 arrived! These will be used to amplify the Pick up backbone with overlaps for the full-length Cdi operon. PCR should yield a 3391 bp fragment.
PCR
PCR Mix 1
GXL buffer
10 uL
dNTPs (from GXL kit
4 uL
GXL polymerase
2uL
ICD0014 (10 uM)
1 uL
ICD0015 (10 uM)
1uL
1:10 diluted plasmid X
1 uL
ddH2O
31 uL
Program
time
Start cycle (30x)
98 °C
10 s
60 °C
15 s
68 °C
35 s
close cycle
8 °C
store
Turned out that the template was wrong. Inoculated 3 ml LB+chloramphenicol for overnight culture of correct Curly clone (clone No. 3) for Miniprep tomorrow.
15/05/22
PCR, Gibson Assembly and Transformation
Miniprep of 1.5 ml of Curly clone No. 3 according to Thermo Scientific Protocol.
PCR with primers ICD014 and ICD015 and freshly prepped Curly plasmid to amplify the backbone for the CDI plasmid (pICD001).
Reaction mix and PCR program see 15-05-21.
Analytical gel: Correct size! (3391 bp)
Figure 1:
Ladder, - , PCR
Proceeded with PCR clean up with subsequent Nanodrop measurement:
Gibson assembly of Curly plasmid/ICD014+015 PCR fragment and CDI operon (EC93 genomic DNA with ICD001 +008)
G.A. reaction mix:
Gibson mix
volume
Insert
2.65ul (= 151ng = 0.02 pmol)
Backbone
0.54ul (= 45ng = 0.02 pmol)
G.A. master mix
10 uL
ddH2O
6.81 uL
G.A program:
temperature
time
50 °C
15 min
16 °C
store
Electroporation of Gibson assembly mix with NEB turbo
• Thaw NEB turbo cells on ice
• Dilute G.A. mix 1:3 (5ul mix + 10ul ddH2O)
• Add 2ul of dilution to 25ul of NEB turbo cells
• Transfer the cells to electroporation cuvette
• Electroporate and add 975ul SOC medium immediately
• Recover 1h at 37°C
• Spin down briefly and discard supernatant
• Plate on chloramphenicol plate
15/05/26
PCR, Gibson Assembly and Transformation
On 15-05-22 DpnI digestion has been forgotten, so the transformed cells probably carry the Curly plasmid instead of the Gibson assembly product. So we repeated the procedure including DpnI digestion.
PCR of Curly backbone: See 15-05-21
DpnI digestion program:
temperature
time
37 °C
30 min
16 °C
store
(No enzyme deactivation was carried out)
Gibson assembly of Curly plasmid/ICD014+015 PCR fragment and CDI operon (EC93 genomic DNA with ICD001 +008)
G.A. reaction mix:
Gibson mix
volume
Insert
2.65ul (= 151ng = 0.02 pmol)
Backbone
0.51ul (= 45ng = 0.02 pmol)
G.A. master mix
10 uL
ddH2O
6.84 uL
G.A program:
temperature
time
50 °C
15 min
16 °C
store
Electroporation of Gibson assembly mix with NEB turbo
• Thaw NEB turbo cells on ice
• Add 2ul of Gibson assembly product to25ul of NEB turbo cells
• Transfer the cells to electroporation cuvette
• Electroporate and add 975ul SOC medium immediately
• Recover 1h at 37°C
• Spin down briefly and discard supernatant
• Plate on chloramphenicol plate (300µL of 0,4% Glucose added to the plate to reduce transcription of Lac promoter)
Gibson mix was not diluted 1:3 in contrast to first attempt on 15-05-22
15/05/27
Plasmidisolation
Colonies are grown wich hopefully carry CDI plasmid. To check this, 12 colonies have been picked and cultivated in 3mL LB broth with additional 0.4% glucose.
In the evening, 1.5ml of cell suspension were pelleted and Miniprep was carried out with the Qiagen Robot.
15/05/28
Plasmidisolation, SacI Digest
The concentration of all the 12 colonies was measured and noted as follows:
plasmid
concentration ng/uL
#1
77
#2
30
#3
90
#4
88
#5
88
#6
86
#7
97
#8
74
#9
84
#10
88
#11
62
#12
81
A mastermix for all plasmids was made:
Digest
volume
DNA template
10 uL
16 °C
store
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