Team:HK SSC/Notebook
17 July
Transformation of dCas9 plasmid 2019 kit plate 5 well 22N.
- Kanamycin Resistance plate
- to E. Coli Neb 5a
19 July
Picked 6 colonies
-Grow overnight in LB
Plasmid Purification (Takara)
-Colony 1 & 2 did not grow
-Run agarose gel 100V 30 mins
-Eluted DNA using 30uL elution buffer
-Nanodrop
22 July
Plasmid prep
Run gel for dcas9
Resuspend dna
23 July
1) Assembling frag1 +2 +3
-RE cut + ligation
NheI MliI BssSI-v2
2) dcas9 preparation Clean
3) Run gel Anson 5α
Transformed produce to Ecoli NEB5a
4) Cloned GFP to dCas9
-RE cut + ligation
EcoRI NsiI
Transformed product to Ecoli NEB5a
5) Transformed PUC19 from stock (for cloning sgRNA later)
1 Aug
Cyanobac arrive, cultured in 15x4 mL medium
2 Aug
6 Aug
Colony PCR
PCR Clean, RE ligation
7 Aug
-gel purification after 75V 45min 0.7 gel
- Gibson assembly
- Run Gel for Overnigt SgRNA PCR product
- RE(2hrs incubation)+ligation for SgRNA and pETBlue
-Transformed 3in1, pETBlue sgRNA and L3 into NEB5a
8 Aug
Colony PCR, run gel, pick clone
9 August
1) Checked plates for sgRNA pet blue mcyI
sgRNA pet blue mcyI 2
sgRNA pet blue mcyB 2
3 in 1
Gib 2.1
Gib 2.2
-All no colony.
2) Conducted PCR purification of overnight (14h) RE cut of dCas9 GFP
-50rxn mixture
3) Conducted ligation
1:3 & 1:5
Vector 250ng
Both 20uL rxn,
-rm temp for 10 min
Heat inactive at 65°C for 10 min
On ice 90min
-Then add ligase again
Ligate over weekend.
4) Amplified frag1 + 2 + 3
-sgRNA I, sgRNA B, GFP
For Gibson
Frag 1 2 3 gfp, 65°C
sgRNAs 55C
All 50 uL rxn
5) Weight powder, for SOC (800mL no glucose/ mgXX) (no yellow)
-LB agar (have yellow) 1800uL
Glucose 9g
6) Run gel for all 6 frags (all in)
-75V 45 mins
Ladder use 1kb & 50bp
Loading dye 6X
7) Gel Purification -elude using 30uL elution b.
12 August
1) Colony PCR for those without bands reaction
-Picked colonies from master plate
2) Performed Gibson(Gib3) & NEBuilder
- Scaled down to 10mL rxn
- Concentration acc to following nanodrop
-incubation 1h
3) PCR for XGib 1 (frag1) Gib1 (frag1)
XGib 2 (frag2) Gib2 (frag2)
XGib 3 (frag3) Gib3 (frag3)
4) Colony PCR no.24 correct band
(dcas9 gfp) L3
Band size 700-800 bp
Inoculated 24 (picked from master plate)
5) 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
1) Run gel for previous PCR
2) Checked plates from 12/8
Colony: petblue +ve
dCas9 gfp 1:3
dCas9 gfp 1:5
3) Plasmid purification L3
dCas9 gfp 24
Re Cut for frag1-3 (gib)
McyI, McyB
14 August
1) Run Gel for X gib Frag 1
-No bands at all
Possibly due to pipetting errors.
2) Started 10uL reaction using Gibson concentration according to following gel. (pic Gib4)
Reaction: Frag3: 50ng
Frag2: 38.73ng
Frag1: 29.26ng
Nebuilder: 5uL
H2O
3) Set up PCR reaction for XGib Frag2
XGib Frag3
| 98°C | 98°C | 66° | 72°C | 72°C | 4°C |
| 30sec | 5s | 15s | 1.5min | 2min | ¥ |
4) Transformed Gib 4.1 Gib 4.2
Overnight ligation sgRNA mcyI Petblue
sgRNA mcyB petblue
Gib -0.9uL DNA
sgRNA -2uL
5) Checked plates for previous transformation: Gib 3 in 1 -
Gib 2 - No colonies
Gib 3 -
From last yr -petblue stock -yes colonies
6) Picked 6 colonies from petblue
Petblue 1-3 -3mL (with
Petblue 4-5 -2mL amp)
Master plate -No xgal Iptg
7) Run gel for pcr products
Frag 2, frag 3 X Gib
Gel purification
DNA concentration on 13/8
15 August
1) RE cut for frag1 frag 2 and frag 3
X Gib ~6 hr
Nanodrop before RE cut
2) Set up Gibson reaction
a. Frag 3 50nL 16uL -
b. Frag 2 38.73ng 2.1 uL -
c. Frag 1 29.26ng 0.4uL - 20uL/xh
d. Nebuilder 10uL -
e. H2O 5.9uL -
50C for 2hr
3) Plasmid Purification of Petblue
Elude using 30uL
Nanodrop
4) Gib 4.1 -Petblue mcyB plasmid have 1 colony each
-Picked & inoculated
5) PCR purified RE cuts from (nolning?)
-ligated H3 & 3 in 1
1+3 1:1
1+2+3 1:1:1
6) Sequencing
16 August
1) Plasmid purification of
Gib4.1 & pET blue mcyB plasmid from 1518
Gib4.1 does not seem to grow
During inoculation
Therefore, low yield.
2) Run gel for frag 1+3 & 3in1 (ligated)
3) Intended to amp 1+3 using 3F + 1R and 3R + 1F
4) Intended to check Gib4 using 1+2, 2+3, 1+3 primers.
(3) and (4) failed possibily due to pcr errors
-Use 3 mins extension 65C other times and maximum
5) Intended to check sgRNA mcyB petblue using colony PCR
(failed due to 0.7% gel, not 1.5% gel pic in chelsy’s phone
19 August
1) Colony PCR for McyB sgRNA petblue
(only 1 white colony)
Results positive: band size ~300bp
2) Inoculated 2 3mL tubes of Gib4
-Gib4.1 & 4.2
From master plate
3) Set up overnight PCR to amp mcyI, frag1, frag3 xgib,
4) RE cut mcyI (247.8ng), petblue (1000ng), frag1 (741ng)- frag3 (140ng) overnight [McyI only added 3uL]
5) Checked Neb 3 in 1 using different primers
Checked frag 1+3
20 August
1) Run gel for mcyI, frag1-3 xGib
-gel purification Takara
2) PCR clean mcyI, petblue 2 from overnight RE
3) Ligated mcyI petblue, frag1+3
-vector 100ng -vector 100ng
1:3 1:1
4) ?
5) Plasmid pur Gib6.4
-yield extremely low
6) Picked colonies: 2 Nebuilder 3in1. -
2 Gib4 - Master Plate
2 sgRNA mcyB petblue -
21 August
1) Plasmid pur for 6 tubes -NEbuilder x2, gib4 x2, sgRNA mcyB petblue
2) Collected overnight ligation
-Amplified frag 1+3 wing
Primer frag1 F & primer frag3 R (vice versa also)
3) Transformed sgRNA mcyI petblue and dCas9 gfp (L3)
4) Received sanger sequence results
-99.4% identical
Mutation- X
5) Picked 25 colonies from previous L3 plate
-colony PCR 25-49 -one Taq
50 - sapphire
6) Single cut & double cut 3 in 1 plasmid
-All undesired results
22aug
Gibson assembly ,incubate 2h
Run gel for colony pcr and frag 1+3 and the overnight cut of 3 in 1
For colonies with correct band after colony pcr, pick colony from master plate, inoculate
Frag 1+3, frag 2 re cut,
Send mcyB to sequencing
pick colony from plates if have
Transform gibson products
Ligate frag 2 to 1+3
23 aug
Run gel for Pcr dcas9 gfp,
Run gel for Pcr 1+3 sgrna mcyI pet blue
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 pur using traditional method
Re cut gib 5
28 aug
PCR screening mcyI, B
29 aug
Plasmid pur. Using tradition n takara
Inoculate gib 5, prepare for measurement
30 aug
I think no lab
31 aug
Pick sgRNA clone?
1/9
I think no lab
2/9
I think no lab
3/9
Re cut dcas9 gfp
4/9
Run gel for re cut dcas9 gfp
5/9
Transformation inoculate pet blue, 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 re cuts
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 re cuts from 37C n run gel (1.5 gel 65V 45 min) shd be ~5500 and ~3500
11/9
PCR clean for 3 in 1 cut
Run gel for the dcas9 gfp sgrna no. 3 re cut for dcas9 gfp sgrna, cut the 5500 band (65V 45 min) (big well) (all in)
Gel pur for the above cut gel (GM buffer 650ul)
Run gel for colony pcr yesterday (1.5 gel)
Set ligation mixture (re 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 RE of 3 in 1 and sgrna dcas9 gfp
13/9
Measure OD (growth curve) for modelling
16/9
Re cuts for final product
pick colony for growth curve
17/9
growth curve
Run gel for the RE cuts, pcr clean, start ligation
transform ligation mixture
18/9
Transofrmation of final ligation products
2. OD measurements
19/9
Pick colony (fat Clone)
Transformation final product
20/9
Pick clone
Pcr check band
· Correct
Plasmid pur by wch failed cuz me filp his gel..
23/9
pick colony of final product
24/9
25/9
Nanodrop final product
26/9
27/9
run gel for the RE cuts and final product 24 and 15.
All in for re cuts,
5ul for final product 24 and 15
65V 50min
Nanodrop final product
30/9
transformation
final product plasmid 15 17 24 29(BL21)
1/10
2/10
3/10
observe green fluorescence by uv exposure
inoculate
observe green fluorescence again by uv exposure
· All negative
4/10
7/10
8/10
Prepared 0.1mM NaCl, 0.1mM HEPES, 0.1mM Vitb12, all not sterilized. Picked 10 colonies, inoculated in 20mL Lb
10/10
Plasmid purification with ethanol
Filter b12
11/10
OD (growth curve)
Transformation of microcystis
Electroporation
Ligation by wch
Dna purification
16/10
Pour plate with kanamycin resistance and chl
Plasmid Purification of Insert 1 and Insert 2
18/10
Pick Insert 1 and 2 ,BL21 and NEB5a clones
Missing
1. 20-21/7
2. 23/7-8/8
3. 24/9 (no gel pic n whatsapp)
4. 26/9 (no gel pic n whatsapp)
5. 1/10 (no gel pic n whatsapp)
6. 2/10(no gel pic n whatsapp)
7. 4/10(no gel pic n whatsapp)
8. 7/10(no gel pic n whatsapp)