Difference between revisions of "Team:IIT Kharagpur/Notebook"

• LB media prepared (500 ml), autoclaved and stored at 4 degrees.
• 250 ml of LB agar prepared and poured into the plates (chloramphenicol antibiotic used- concentration 1 micro litre/ml)

• DNA from wells dissolved into 10 μl distilled water. DNA kit plate instructions
• Transformed 4μl of DNA into 100μl of E.Coli DH5ɑ. transformation procedure)
• 100μl of transformed cells were plated on chloramphenicol resistant plates.and left overnight to grow.

• Colonies for luxPR promoter (BBa_R6002) constructs were observed. A colony was picked and inoculated in 3 ml culture for glycerol stock preparation.
• No colony observed for luxR protein (BBa_K546003) construct.

• Transformation repeated for luxR protein construct .transformation procedure
• Culture prepared for plasmid isolation for luxPR promoter. Colony was picked and inoculated in 10 ml culture.

• No colony observed for luxR protein construct
• Plasmid isolation done for luxPR promoter
• Gel electrophoresis done for luxPR promoter

• Lane 7 : 1kb marker
  Lane 8 : luxPR promoter
• Gel electrophoresis done for luxPR promoter

	Amount Used	Amount	A 260/280	A 260/230
P1 (luxPR promoter) (BBa_R6002)	1 μl	117.2ng/μl	1.85	1.54

• Cells containing LuxR were inoculated in 10 ml culture and kept overnight.

• LuxR DNA was extracted following the protocol of H1 Media.
• Gel electrophoresis done for LuxR DNA
• Nanodrop done for LuxR.

	Amount Used	Amount	A 260/280	A 260/230
P3 (LuxR) (BBa_K546003)	1μl	191.2ng/μl	1.91	2.14

	Amount Used	Amount	A 260/280	A 260/230
P3 (LuxR) (BBa_K546003)	1μl	191.2ng/μl	1.91	2.14

• Restriction digestion mix. used for LuxR DNA extracted before. Restriction enzymes used:
  1. Xba I
  2. Pst I

Reaction Mixture
DNA	10μl
Buffer	2μl (3.1)
Pst 1	1μl
Xba 1	1μl
H2O	6μl
Kept at 37 degree Centigrade overnight.

• To check the growth of crt EBI (reporter) in Ampicillin + LB , 3ml culture grown overnight.

• Glycerol stocks made from 3 ml culture of crt EBI reporter which was grown overnight. (20% glycerol stock kept in 20℃)
• Gel run for restriction digestion of LuxR.
• Inoculation and streaking done for crt EBI reporter.

• Plasmid preparation for crt easeInOutCubic
• Precautions:
  1. Use only one column for 10 ml culture while extracting.
  2. After loading the supernatant in the column, wait for 10 min for better DNA binding.
  3. Before eluting with (distilled) autoclaved water wait for 10 min. for better elution
• Nanodrop for crt EBI

• 50 ml culture used for extracting luxPR plasmid using E. coli DH5ɑ strain
• 34 μl/ml of antibiotic (camR) used as a resistance marker
• Plasmid extracted for the LuxPR part from 50 ml culture. 60 μl volume of plasmid obtained.

	Amount Used	Amount	A 260/280	A 260/230
P1 (LuxPR) (BBa_R6002)	1μl	170 ng/μl	1.83	2.05

• Restriction digestion of LuxPR and crt EBI

	LuxPR	crtEBI
DNA	8μl	6μl
Enzyme 1	1 μl (Spe I)	1μl (Xba I)
Enzyme 2	1 μl (Pst I)	1μl (Pst I)
Buffer	2 μl (2.1)	2μl (3.1)
H2O	8μl	10μl

• Gel was run with crtEBI containing digested plasmid and LuxPR containing digested plasmid
  Length of crtEBI ~ 3.3 kbp
  Length of LuxPR ~ 50 bp
  Plasmid containing LuxPR ~ 2.2 kbp
• crtEBI part , and LuxPR containing part extracted from gel . DNA eluted using the kit
• Nanodrop was run

	Amount used	amount
LuxPR	1μl	28 ng/μl
crtEBI	1μl	50 ng/μl

• Ligation performed of crtEBI part into plasmid backbone containing LuxPR.
  Insert: crEBI (3.3 kb) , 50 ng/μl
  Vector: LuxPR (2.1 kb) , 28 ng/μl

• Transformed the ligation product of LuxPR and crtEBI(reporter gene). transformation procedure
• Plating done for the above construct.

• No growth observed for the construct. Steps repeated.
• Restriction digestion of LuxPR: (single digestion)

LuxPR	7 μl
Spe I	1 μl
Buffer (cutsmart)	2 μl
H2O	10 μl
Kept at 37℃ for 5-6 hours.

• Second digestion setup made.

Product from previous digestion	20 μl
Pst I	1 μl
Buffer (3.1)	2 μl
Kept at 37℃ for 5-6 hours.

• Gel was run for the restriction digest product.
• Band was cut and DNA eluted using gel extraction kit.
• Ligation set up after purifying the DNA.

• Transformation done for ligated product. Transformation procedure

• A single colony was observed.
• The colony was inoculated into a 3 ml Chloramphenicol containing medium.

• Plasmid extraction done for the colony.
• Screening done by running Gel. The band obtained was below 2 kb size.( Expected band size was 5.4 kb)

• Double digestion performed with Spe I and Pst I.

Reaction Mixture

DNA + Water 20 μl	20 μl
Spe I	0.5 μl
Pst I	1.0 μl
Buffer (2:1)	2.0 μl

• Digested Lux PR eluted from gel.
• Plasmid extraction done for Lux PR. Concentration: 126.5 ng/ μl
  Volume: 55 μl
• Ligation reaction mixture for Lux PR (27 ng/μl) + crtEBI (28 ng/μl)

Reaction Mixture

	V:I = 3:1	V:I=2:1
crtBI (28.2 ng/μl)	3μl	4μl
luxPR (27.2 ng/μl)	6μl	6μl
T DNA ligase	1.5μl	1.μl
ligase buffer	1.5μl	1.5μl
Water	8μl	7μl

ligation @ 16 for 16 hours.

crtEBI	3μl
luxPR	6μl
buffer	1.5μl
ligase	1.2μl
water	3.3μl

Digestion: LuxPR (E and S) ­ 3μl CrtEBI (E and X) ­ 1.4 μl

DNA	12μl
EcoRI	1μl
Buffer	2μl
H2O	5μl

Sequential

DNA	10μl
EcoRI	1μl
Buffer [Neb buffer EcoRI]	2μl
H2O	7μl

Digestion ­ 2

LuxPR	17μl
Spe I	1μl
Buffer (Cutsmart)	2μl

CrtEBI	17μl
Xba I	1μl
Buffer (Cutsmart)	2μl

Result:
2% gel was prepared since the LuxPR insert was only 55 bp. The gel wasn’t prepared properly. The same bands were observed twice. This was probably because the concentration was not uniform throughout the height of the gel. The bands ran faster at the upper surface and slower at the lower surface. The 55 bp was not observed at all. 3 A Assembly
Restriction Digestion:
FIRST DIGESTION
Part A

LuxPR (128 ng/μl)	3μl
Buffer (EcoRI Buffer)	2μl
EcoRI	1μl
N.F./ dd H2O	14μl

Part B

CrtEBI (140 ng/μl )	3μl
Buffer (2:1)	2μl
Xba I	1μl
N.F. H2O	14μl

A and B kept at 37 for 2 hours
Then PCR Purified.
Backbone

DNA	5μl
EcoRI	0.5μl
Pst I	0.5μl
Dpa I	0.5μl
Buffer (2:1)	2μl
N.F. H2O	11.5μl

Kept at 37 for 1 hour
PCR Purification
SECOND DIGESTION

DNA (LuxPR)	20μl
Spe I	1μl
Buffer (Cutsmart)	2μl

DNA (CrtEBI)	20μl
Pst I	1μl
Buffer (3:1)	2μl

Nanodrop data:
LuxPR ­ 15 ng/μl
CrtEBI ­ 15 ng/μl
Backbone ­ 5 ng/μl
Size of plasmids:
LuxPR ­ 2.1
CrtEBI ­ 5.5
Backbone ­ 2.2
Ligation Mixture:
Molar Ratio­­­ LuxPR : CrtEBI : Backbone = 1 : 1 : 0.5 (Recommended)

LuxPR	3μl
CrtEBI	8μl
Backbone	5μl
Buffer	2μl
Ligase (T4)	1μl

Molar Ratio­­­ LuxPR : CrtEBI : Backbone = 3 : 1 : 0.5

LuxPR	6μl
CrtEBI	5μl
Backbone	3μl
Buffer	2μl
Ligase (T4)	1μl
Water (T4)	3μl

3A assembly possible plasmids and their sizes for screening correct plasmid

1-10 b → 1:1:0.5 1-10 a→ 3:1:0.5 7b colony→ plasmid check (68 ng/ μl) CLONE CONFIRMATION
Digestion 1

DNA (68 ng)	7μl
Buffer (2:1)	2μl
EcoRI	0.5μl
Pst I	0.5μl
H2O	10μl

Digestion 2

DNA	7μl
Buffer (CS)	2μl
Xba I	0.5μl
Spe I	0.5μl
H2O	10μl

DNA	7μl
Buffer (3:1)	2μl
Pst I	1μl
H2O	10μl
Total	20μl

3A Assembly (Attempt 4)

LuxR	7μl
EcoRI	1μl
Buffer (EcoRI)	2μl
dd H2O	10μl

CrtEBI	10μl
Xba I	1μl
Pst I	2μl
dBuffer (3:1)	10μl
dd H2O	10μl

Digestion 2

DNA	7μl
Buffer (CS)	2μl
Xba I	0.5μl
Spe I	0.5μl
H2O	10μl

DNA	7μl
Buffer (3:1)	2μl
Pst I	1μl
H2O	10μl
Total	20μl

DNA	13ul
Spe 1	1µl
Pst 1	1µl
Buffer	2ul
dd H2O	3ul

k

DNA(7b)	8µl
Buffer (EcoR1)	2µl
dd H20	9µl

DNA (CrtEBI)	10µl
Buffer (EcorR1)	2µl
dd H2O	7µl

(To get the backbone)

DNA (LuxR)	10µl
Buffer (EcorR1)	2µl
EcorR1	1µl
dd H2O	7µl

#Pst digestion : #1μl Pst 2μl something #Kept at 37℃ for 6 hours.

7b : 12 ng/μl LuxR : 12 ng/μl LuxPR : 20 ng/μl CrtEBI : 20 ng/μl Ligation 1 (standard assembly)

(X/P) CrtEBI (3.3)	7µl
(S/P) LuxPR (2.1)	5µl
T4 Ligase	1µl
Ligase Buffer	2µl

Ligation 2 (breakthrough standard assembly)

(X/P) CrtEBI (3.3)	5µl
(S/P) Lux R (3.3)	8µl
T4 Ligase	1µl
Buffer	2µl