notebook
Jun 2016
01/07/16
JQA
Preparation of dCas9 plasmid: hym12, hym5, jqa2.
03/07/16
JQA
Transformation of Recombinant of sgRNA, sgNC(negative control). In total 6 plates
06/07/16
JZR
Identification of Positive Recombinant of sgRNA, sgNC. And inculated 2 single colony of transformed bacteria
into LB medium. Shaking overnight at 37℃. Transformation of Recombinant of sgActin.
07/07/16
JZR
Identification of Positive Recombinant of sgActin. And inculated 2 single colony of transformed bacteria into
LB medium. Shaking overnight at 37℃.
08/07/16
JZR
Prepare plasmids of sgActin and sgNC with the Kittool. Sent to sequencing.
09/07/16
JZR
PCR for sgActin with KOD system.
PCR system:
- Template 1µl
- Primer F & R 1.5 * 2 = 3µl
- Buffer 5 µl
- dNTP 5 µl
- Mg2+ 4 µl
- KOD+ enzyme 1 µl
- ddH2O 31 µl
total 50 µl
The result of agarose gel electrophoresis is smeared.
10/07/16
GCH,LMJ
In vitro transcription of sgRNA using the promega kittool.The result is negative.
Isolation of total RNA and cDNA synthesis. KOD PCR for linear DNA with T7 promoter.
PCR system:
- Template 1µl
- Primer F & R 1.5 * 2 = 3µl
- Buffer 5 µl
- dNTP 5 µl
- Mg2+ 4 µl
- KOD+ enzyme 1 µl
- ddH2O 31 µl
total 50 µl
11/07/16
LMJ, GCH
In vitro transcription of sgRNA using the promega kittool. RNA purification.
Reaction system:
- 0.1 Volume of NaOAC
- 1 Volume phenol-chloroform-isopropanol
Mix(invert 6 times)
Transfer to phase lock gel(PLG)
15000 rpm, 10min, 4℃
supernatant + same volume of chloroform
Mix
PLG
Transfer to phase lock gel(PLG)
15000 rpm, 10min, 4℃
Add 1.5 volume of isopropanol
-20$#8451 for 1 hour
18000rpm, 4℃
200 µl 75% EtOH wash
5 µl ddH2O resolve
18/07/16
LY
KOD PCR for sgActin & yActin linear DNA with T7 promoter
PCR system:
- Template 1µl
- Primer F & R 1.5 * 2 = 3µl
- Buffer 5 µl
- dNTP 5 µl
- Mg2+ 4 µl
- KOD+ enzyme 1 µl
- ddH2O 31 µl
- total 50 µl
PCR procedure:
- 94℃ 3’
- 94$℃ 30’’
- 60$℃ 30’’ * 25 cycles
- 72$℃ 30’’ * 25 cycles
- 72$℃ 7’
- 16$℃ ∞
PCR result: sgActin failed. yActin succeeded.
19/07/16
LY
sgActin1-4 were sent to sequencing.(M13F)
PCR of sgActin, procedure is the same as that in 7.18
sgActin PCR purification using TIANGEN Kit.
The result of agarose gel ep is negative. – Nothing!
28/07/16
JQA
dCas9 protein
A280 test suggests a 0.62 ng/ml
28/07/16
LMJ
dCas9 +RT+sgRNA transformation into yeast
dCas9 +PAMMer+sgRNA transformation into yeast
using leu2 auxotroph yeast to select positive strains.
29/07/16
JQA
Bridge PCR of Actin with 3 parallels.
Colony PCR of RT with KOD(20 µl system)
Reaction buffer:
- 20 mM Tris-HCL pH7.5
- 75mM KCl
- 5mM MgCl2
- 1mM DTT
- 5% glycerol
Aug 2016
01/08/16
GCH, LMJ
POT assembly system:
- 10X T4 ligase buffer 1µl
- 100X BSA 0.1µl
- BsmBI 0.5 µl
- T4 ligase 0.2µl
- Parts: P,O,T 2µl
- POT vectors
In vitro transcription with promega Kit (20 µl system):
- Buffer 4µl
- ATP, CTP, GTP, UTP: 1.5µl*4=6µl
- Enzyme mix(T7) 2µl
- Template: 1-2 µg
- Nuclease free water 7µl
37$#8451 incubation for 2hrs
DNAase overnight
PCI extraction
EtOH precipitation
Free water resolve
Agarose gel to confirm
02/08/16
LMJ
SgRNA extraction
SgRNA template PCR purification
03/08/16
JQA
POT assembly of RT, yTK
Couldn't find 555,556, so did plasmid preparation again
20/08/16
JQA
C2C2 assembly:
PCR system:
- C2C2 + primer
- dCas9 POT + BD primer/AD primer
- Template 5µl
- Primer F & R 2.5 * 2 = 3µl
- Buffer 5 µl
- dNTP 5 µl
- Mg2+ 4 µl
- KOD+ enzyme 1 µl
- ddH2O 25 µl
total 50 µl
PCR procedure:
- 94℃ 3’
- 94℃ 30’’
- 55℃ 30’’ * 25 cycles
- 68℃ 4’ * 25 cycles
- 68℃ 10’
- 16℃ ∞
21/08/16
JQA
BD/AD PCR failed
Using PID 13, 14 to restart the experiment again.
PCR system:
- Template 5µl
- Primer F & R 2.5 * 2 = 5µl
- Buffer 5 µl
- dNTP 5 µl
- Mg2+ 4 µl
- KOD+ enzyme 1 µl
- ddH2O 31 µl
total 50 µl
PCR procedure:
- 94℃ 3’
- 94℃ 30’’
- 55℃ 30’’ * 35 cycles
- 68℃ 345’ * 35 cycles
- 68℃ 10’
- 16℃ ∞
23/08/16
JQA
One pot digestion & ligation reaction to construct dC2C2-BD-AD on HCKanO
KOD PCR to change vector for dCas9
24/08/16
LY
Construct PQlink-His-dCas9
PQ-H double digestion:
- 10X NEB buffer 3.1 2µl
- EagI 0.5µl
- SalI-HF 0.5µl
- ddH2O 13µl
Total 20µl
37℃ 2hrs and 65℃ 20’
27/08/16
LMJ
PCR + Histag to dCas9
PCR system:
- Template 5µl
- Primer F & R 2 * 2 = 4µl
- Buffer 5 µl
- dNTP 5 µl
- Mg2+ 4 µl
- KOD+ enzyme 1 µl
- ddH2O 25 µl
total 50 µl
PCR procedure:
- 94℃ 3’
- 94℃ 30’’
- 63℃ 30’’ * 25 cycles
- 68℃ 6’ * 25 cycles
- 68℃ 10’
- 16 ∞
Ligation
Transformation into
E.coli
Shaking overnight
30/08/16
JQA
C2C2 assembly:
Result is negative again!
31/08/16
JZR
Using fluorescence to locate dCas9, via electroporation
Sg+ group: sg+ Cas9+PAMer
Sg- group: Cas9+PAMer
Most signal is located in the nucleus
Sep 2016
04/09/16
LMJ
Transformation of pGEX-suvCas9 plasmid
05/09/16
LY
C2C2 assembly again
BD, AD, C1C2 gel clean up digestion
- DNA 4µl
- Custmart 1µl
- BsaI 0.2µl
- ddH2O 4.8µl
06/09/16
LY
C1-C2, BD-AD PCR
- Q5 buffer 5µl
- dNTP 4µl
- Primer 2.5* 2µl
- Q5 0.5µl
- Ligation product 30.5µl
18/09/16
LY
Transform BL21 with pGEX-suvCas9
Inculate in LB, 37℃ for 8hrs
23/09/16
JZR
Insert sequencing
DNA purification in 15µl system
Nanodrop to measure the concentration:
- Insert5-1: 3.6λ
- Insert12-1: 8.7λ
24/09/16
JZR
Transfection with yeast using 2 system:
system1 :insert5-1
system2 :insert12-1
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