Difference between revisions of "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)