Nidhi: Creating an N-terminal fusion of sax-2 gene short isoform.
a)
Created the sgRNA::CAS9 construct (pJW1219, 8168bp) containing the
gRNA corresponding to sax-2 gene short isoform via SDM.
b)
RE digestion of repair template (pDD282_GFP_isolate 3, 10475bp)
with AvrII and SpeI.
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Debjani: Creating a C-terminal fusion of unc-11 gene knock in.
Forward
primer : cgactagaCTAtaatccaaaGTTTAAGAGCTATGCTGGAA
Reverse
Primer : CAAGACATCTCGCAATAGGA
Purpose : For SDM to
create the Cas9-sgRNA construct (pJW1219) to include the guide RNA (no PAM) corresponding to unc-11 knock in.
Restriction
enzyme digestion of repair template (pDD286_RFP_Knock in ) with AvrII and NgoMIV.
---------------------------------
Leena: Ran a 1% agarose gel
with 2-log DNA ladder(0.1-1kb)
Creating a C-terminal fusion for sax-2 gene, using NEB Q5 Site-Directed Mutagenesis. We are using Cas9-sgRNA construct (pJW1219-10, 8168bp) containing target sequence(gRNA) to knock-in target sequence of 20 nucleotides. Simultaneously creating a repair template by doing restriction digestion of ccdb in pDD286-6 using AvrII and NgoMIV.
Verifying the following:
1.RE digestion of repair
template(pDD286-6) removing ccdb using AvrII and NgoMIV - 5ul sample was
loaded
MLP-RE
DNA: pDD286-6 (10504 bp),
221.0ng/ul- 4.5ul
R.Enzymes:
AvrII,5U/ul – 2ul
NgoMIV,10U/ul- 1ul
RE digest products: 6654 bp,
2638 bp, (609 bp,603 bp – as one band)
2. PCR amplification of sgRNA::CAS9 construct (pJW1219-10, 8168bp) containing the target sequence, knock in - C-terminal fusion using SDM. - 4ul sample was loaded
Gene: Sax-2
MLP-PCR : Project-4, Sax-2 C-term
fusion
DNA: pJW1216 (8148 bp)
(25ng/ul) -0.75ul
10uM Forward primer:
MG_2215_gRNA_sac2_C-2 – 0.94ul
10uM Reverse primer:
MG_2203_gRNA_R_6141- 0.94ul
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Jose: Project 4-C-terminal sax-2 knock-in
Creating the Cas9-sgRNA construct (pJW1219, 8168bp) containing target sequence(gRNA) using NEB Q5 Site-Directed Mutagenesis to knock-in target sequence at the C-terminal region on Sax-2 gene.
Restriction enzyme double digest was done using PDD282_GFP_Repair Template (isolate 3) using enzymes AvrII and SpeI.
We used 1% Agarose Gel with NEB 2-Log DNA ladder (0.1-1kb).
Jose: Project 4-C-terminal sax-2 knock-in
Creating the Cas9-sgRNA construct (pJW1219, 8168bp) containing target sequence(gRNA) using NEB Q5 Site-Directed Mutagenesis to knock-in target sequence at the C-terminal region on Sax-2 gene.
Restriction enzyme double digest was done using PDD282_GFP_Repair Template (isolate 3) using enzymes AvrII and SpeI.
We used 1% Agarose Gel with NEB 2-Log DNA ladder (0.1-1kb).
Names
|
SDM
Code
|
Primer Codes
|
Gene
|
Expected RE Digest Bands
|
Summary of Transformation Results(ng/μL)
|
Reverse primer for SDM-PCR:
Used by all of us
MG2203_sgRNA_R_6141
| |||||
Jose
|
JAH-15
|
MG2210 _gRNA_sax-2_C
|
Sax-2
|
(PDD282_GFP)
6,625bp and 2,643bp(SpeI), & two 603 bp bands(AvrII)
|
Plasmid DNA concentrations
JAH-18-1: 572.5
JAH-18-2: 403.8
JAH-18-3: 376.3
JAH-18-4: 428.4
|
Leena
|
MLP21
|
MG2215_gRNA_sax-2_c-2
|
Sax-2
|
pDD286-6,digested with AvrII and NgoMIV
Expected and has observed following bands.
6654bp,2638bp,[609,603bp- seen as 1 band]
|
The transformation worked and the following are the plasmid DNA concentration of the single colonies, derived after DNA mini prep, measured concentration using Nanodrop:
MLP-500-1-33: 141.5 ng/ul
MLP-500-2-33:184.0 ng/ul
MLP-50-1-33: 366.7 ng/ul
MLP-50-2-33: 165.6 ng/ul
|
Nidhi
|
ND_10
|
MG_2209
_gRNA_sax-2_N_S
|
sax-2
N terminal, short isoform
|
ND_8
(PDD282_GFP, isolate3, C-terminal fusion Digested with AvrII and SpeI)
Bands:6,626bp, 2,643bp, and two 603 bp
|
ND_16a: 328.1 ng/ul
ND_16b: 203.3 ng/ul
ND_16c: 285.2 ng/ul
|
Debjani
|
DP _13
|
MG2213_gRNA _unc-11_C
|
unc-11 C terminal knock in
|
DP_11
(PDD286_RFP)
C-terminal fusion digested with AvrII and NgoMIV
Bands: 6,628bp, 2,643bp, and two 603 bp
|
Plasmid DNA NanoDrop concentration
DP26_1:462.0 ng/μL,
DP26_2: 347.9ng/μL, DP26_3: 319.3 ng/μL
DP26_4: 327.5 ng/μL
|
