Team:HK SSC/Notebook
17 July
Transformation
of dcas9 plasmid 2019 kit plate 5 well 22N
to E. coli
NEB5a using kanamycin resistance plate
19 July
Picked 6
colonies
grow
overnight in LB
Plasmid
purification (Takara)
colony 1
& 2 did not grow
run agarose gel
for 100v 30 mins
eluted DNA
using 30ul elution buffer
nanodrop the eluted DNA
22 July
Plasmid prep
Run gel for dCas9
Resuspend DNA
23 July
Assembling Fragment 1+2+3
restriction
endonuclease cut + ligation
Using NheI and BssSI-v2
Dcas9
preparation clean
Run gel dCas9
Transformed
produce to E.coli neb5a
Cloned GFP
to dcas9
restriction
endonuclease cut + ligation
Using EcorI NsiI
Transformed
product to E.coli neb5a
Transformed puc19
from stock (for cloning sgrna later)
1 Aug
Cyanobacteria
arrived, cultured in 15x4 ml medium
GFP-dcas9
plasmid ligation (all failed)
2 Aug
Colony PCR for L2, 3, 4
PCR Fragment 1, 2, 3
Plasmid purification
6 Aug
colony PCR
standard q5 PCR
preperation for gibson
Gibson assembly
GFP+ dCas9
plasmid again as L3 Failed
7 Aug
gel
purification after 75v 45min 0.7 gel
gibson assembly
run gel for overnight
sgrna PCR product
restriction
endonuclease cut (PstI, HindIII)(2hrs
incubation)+ligation for sgRNA and PETblue
transformed
3in1, PETblue sgRNA and L3 into NEB5a
8 Aug
Colony PCR
for l3
Run gel for
3in1 (1 smear),
Pick clone
9 August
Checked
plates for sgRNA PETblue McyI(no colony)
sgRNA pet blue McyI(no colony)
sgRNA pet blue Mcyb (no colony)
3 in 1(no colony)
Gib 2.1(no
colony)
Gib 2.2(no colony)
Conducted PCR
purification of overnight (14h) restriction endonuclease cut of dCas9 GFP using
EcoRI, NsiI
50 μL rxn mixture
Conducted
ligation
1:3 &
1:5
Vector 250ng
20 μL rxn mixture
Placed in room
temperature for 10 min
heat
inactive at 65°c for 10 min
on ice 90min
then add
ligase again
ligate over
weekend.
Amplified Fragment1
+ 2 + 3
sgrna McyI, sgRNA
McyB, GFP
Fragment 1 2
3 GFP, 65c
sgRNA 55c
All 50 μL
rxn
Weight
powder, for soc (800ml no glucose) (no yellow)
LB agar
(have yellow) 1800ul
Glucose 9g
Run gel for
all 6 Fragments (all in)
75V 45 mins
Ladder use
1kb & 50bp
Loading dye 6x
Gel purification
-elute using 30ul elution buffer.
12 August
Colony PCR
for those without bands reaction
picked
colonies from master plate
Performed gibson(gib3) & nebuilder
Scaled down
to 10ml rxn
PCR for xgib 1 (Fragment1) gib1 (Fragment1)
xgib 2 (Fragment2) gib2 (Fragment2)
xgib 3 (Fragment3) gib3
(Fragment3)
Colony PCR
no.24 correct band
(dCas9 GFP) L3
band size
700-800 bp
inoculated
24 Hours (picked from master plate)
Transformed gibson 1 gibson 2 gibson 3.2 nebuilder
(1)
+ve control petblue nebuilder (2)
dcas9 gfp
1:3
dcas9 gfp
1:5
gibson 3.1
13 August
Run gel for
previous PCR
Checked
plates from 12/8
Colony: PETblue +ve
dCas9 GFP 1:3
dCas9 GFP 1:5
Plasmid
purification L3
dCas9-GFP 24
Restriction
endonuclease for Fragment1-3 ( for gibson
assembly)
McyI, McyB
14 August
Run gel for xgib Fragment 1
no bands at
all
Possibly due
to pipetting errors.
Started 10ul
reaction using gibson concentration according to
following gel. (pic gib4)
Reaction: Fragment3:
50ng
Fragment2:
38.73ng
Fragment1: 29.26ng
nebuilder: 5ul
h2o
1) Set up PCR reaction for xgib Fragment2
Xgib Fragment3
|
98°c |
98°c |
66°c |
72°c |
72°c |
4°c |
|
30sec |
5sec |
15sec |
1.5min |
2min |
¥ |
2) Transformed gib 4.1 gib 4.2
Overnight
ligation sgrna McyI petblue
Sgrna Mcyb PETblue
gib -0.9ul dna
sgrna -2ul
3) Checked plates for previous
transformation: gib 3 in 1 -
gib2 - no colonierom
last year -PETblue stock - there are colonies
4) Picked 6 colonies from PETblue
PETblue 1-3 -3ml (with amp)
PETblue 4-5 -2ml
Master plate -no xgal iptg
5) Run gel for PCR products
Fragment 2, Fragment 3 xgib
Gel purification
DNA concentration on 13/8
15 August
Restriction
endonuclease cut for Fragment1 Fragment 2 and Fragment 3
Xgib ~6 hr (MluI, BssaI and NheI)
nanodrop
before restriction endonuclease cut
Set up gibson reaction
Fragment 3 50ng
16ul
Fragment 2 38.73ng 2.1 ul
Fragment 1 29.26ng 0.4ul
Nebuilder 10ul
H2o 5.9ul
50°c for 2hr
(optimized protocol)
Plasmid purification
of PETblue
eluted using 30ul elution buffer
nanodrop
Gib 4.1 -PETblue McyB plasmid have 1
colony each
picked & inoculated
PCR purified
restriction endonuclease cuts from (Fragment 1, 2, 3)
ligated h3
& 3 in 1
1+3 1:1
1+2+3 1:1:1
Sequencing
16 August
Plasmid
purification of
Gib4.1 &
pet blue McyB plasmid from 1518
Gib4.1 does
not seem to grow during inoculation therefore, low yield.
Run gel for Fragment
1+3 & 3in1 (ligated)
Intended to
amp 1+3 using 3f + 1r and 3r + 1f
Intended to check
gib4 using 1+2, 2+3, 1+3 primers.
3 and 4
failed possibly due to PCR errors
use 3 mins
extension 65°c other times and maximum
Intended to
check sgRNA Mcyb PETblue
using colony PCR
(failed due
to 0.7% gel)
19 August
Colony PCR
for Mcyb sgRNA PETblue
(only 1
white colony)
Results
positive: band size ~300bp
Inoculated 2
3ml tubes of gib4
gib4.1 &
4.2
From master
plate
Set up
overnight PCR to amp mcyI, Fragment1, Fragment3 xgib,
Restriction
endonuclease (PstI, HindIII,
MluI, BssaI, NheI) cut McyI (247.8ng), PETblue (1000ng), Fragment1 (741ng)- Fragment3 (140ng)
overnight [McyI only added 3ul]
Checked neb
3 in 1 using different primers
Checked Fragment
1+3
20 August
Run gel for mcyI, Fragment1-3 xgib
gel purification
Takara
PCR clean McyI, PETblue 2 from overnight restriction
endonuclease cut
Ligated McyI PETblue, Fragment1+3
vector 100ng -vector 100ng
1:3
1:1
Plasmid purification
gib6.4
yield
extremely low
Picked
colonies: 2 from nebuilder 3in1.
2 from
gib4 -master
plate
2 from sgrRNA McyB PETblue-
21 August
Plasmid purifucation for 6 tubes - nebuilder
x2, gib4 x2, Sgrna Mcyb PETblue
Collected
overnight ligation
amplified Fragment
1+3
Primer Fragment1
f & primer Fragment3 (vice versa also)
Transformed sgRNA
McyI PETblue and dCas9 GFP
(L3)
Received
sanger sequence results
99.4%
identical
Mutation
\ wrong
Picked 25
colonies from previous l3 plate
colony PCR 25-49 -One Taq
50 - Sapphire
Single cut
& double cut 3 in 1 plasmid
all
undesired results
22 Aug
Gibson assembly, incubate 2h
run gel for colony PCR and Fragment
1+3 and the overnight cut of 3 in 1
For colonies with correct band after colony PCR, pick colony from master
plate, inoculate
Fragment 1+3, Fragment 2 re cut,
Send McyB to sequencing
Transform gibson products
Ligate Fragment 2 to 1+3
23 Aug
Run gel for PCR dCas9 GFP,
Run gel for PCR 1+3 sgRNA MCYI PETblue
26 Aug
Run gel for previous 40 colony PCR
Inoculate l3 with correct band size
Preparation for electroporation
Transform ligation product and 3 in 1 for characterization
27 Aug
Pick clone for previous transformation
3 in 1 plasmid purification using ethanol perpetration
Restriction endonuclease cut gib 5 (MluI)
28 Aug
PCR screening McyI, McyB
29 Aug
Plasmid purification. Using Takara
Inoculate gib 5, prepare for measurement
3/9
Restriction endonuclease cut dCas9 GFP (EcoRI,
PstI)
4/9
Run gel for restriction
endonuclease cut dCas9 GFP
5/9
Transformation inoculate PETblue, Puc19, 3 in
1, PsB1c3 transformants
Plasmid purification 3 in 1 using Takara plasmid purification kit
6/9
Plasmid purification 3 in 1 using Takara plasmid purification kit
9/9
2 restriction endonuclease cuts (dCas9-GFP-sgRNA)
10/9
Run gel for the colony PCR products (1.5 gel, all in) 65v 45 min 700 or
no band
Pick colony and colony PCR
Pour agar plate
take the 2 restriction
endonuclease cuts from 37c n run gel (1.5 gel 65v 45 min) shd
be ~5500bp and ~3500bp
11/9
PCR clean for 3 in 1 cut
Run gel for the dcas9 gfp sgrna
no. 3 restriction endonuclease cut for dcas9 gfp sgrna (XbaI, XhoI),
cut the 5500 band (65v 45 min) (big well) (all in)
Gel purification for the above cut gel (gm buffer 650ul)
Run gel for colony PCR yesterday (1.5 gel)
Set ligation mixture (restriction endonuclease cuts of 3 in 1 and mcy
dcas9 gfp) 1:1
12/9
Heat inactivate ligation
2. Transform ligation (total dna 60ng)
3. Dilute overnight inoculation, 1 to 3
4. Set restriction endonuclease cut of 3 in 1 and sgrna
dcas9 gfp
13/9
Measure od (growth curve) for modelling
16/9
Restriction endonuclease cuts for final product (XhoI,
XbaI)
Pick colony for growth curve
17/9
Growth curve
Run gel for the restriction endonuclease cuts, PCR clean, start ligation
Transform ligation mixture
18/9
Transofrmation of final ligation products
Od measurements
19/9
pick colony (fat clone)
Transformation final product
20/9
Pick clone
PCR check band (Correct band at 12kbp)
Plasmid purification (Takara)
23/9
Pick colony of final product
24/9
Plasmid purification (Takara)
25/9
Nanodrop final product
26/9
Restriction
endonuclease cut for final product (PstI)
27/9
Run gel for the restriction endonuclease cuts and final product 24 and
15.
All in for restriction endonuclease cuts,
5ul for final product 24 and 15
65v 50min
Nanodrop final product
30/9
Transformation
final product plasmid 15 17 24
29(BL21)
3/10
Observe green fluorescence by UV exposure
Inoculate
Observe green fluorescence again by UV exposure
All negative
4/10
Plasmid purification of final
product
7/10
Transformation of final products
into DH5a
8/10
Prepared 0.1mm nacl, 0.1mm hepes,
0.1mm vitb12, all not sterilized. Picked 10 colonies (final product) , inoculated in 20ml LB
9/10
Filtered Vitamin B12
Autoclave bold3n
Add to agar
10/10
Plasmid purification with ethanol percipitation
11/10
Measure Optical density (growth curve)
Transformation of Microcystis
Electroporation
Ligation by Peter
DNA purification
14/10
Transformation
of previous ligation
15/10
Pick clones,
inoculate i12
Standard
solution
16/10
Pour plate
with kanamycin resistance and chloramphenicol
Takara
plasmid purification of insert 1 and insert 2
17/10
Peter ethanol precipitation of final product
Electroporation
of Microcystis Aeruginosa UTEX 2388
Ligation
mixture (cut with SpelI, xbaI)
Run gel for
plasmid purification product of I2
Nanodrop I2
Anson adjust
pH for BG11
Error: labelling
error for plasmid purification
18/10
Pick insert
1 and 2 ,BL21 and NEB5a clones