Team:NU Kazakhstan/Notebook
Notebook
1.06.15
- Preparation of the LB agar
We used the 37 g of nutrient agar for 400 mL of the distilled water - Extraction of genome from S.mutans
- First, culture the S.mutans in 5 mL liquid BHI + bacitracin
- Centrifuge at 15 min for 4000 rpm
- Then dissolve the pellet in 500 microliter of lysis buffer
How to prepare Lysis Buffer (1 mL)
Lysis Buffer contains the EDTA, Tween 80%, tris HCl, 125 microliter of 8M EDTA, 5 µl of of Tween 80, Tris HCl 1M 50 µl, Proteinase K (we need 200 µg/mL). We took 0.0002 grams of powder Proteinase K for 1 mL of lysis buffer. The balance could not read 0.0002 grams of Proteinase K, so 0.02 of proteinase K was taken.
4. Incubation for the 2 hours at 55°C. Then there is a need to heat at the 90°C for 5 minutes.
5. Then add equal amount of cold isopropanol.
6. There is a need to incubate in freezer for the 20 minutes.
7. Centrifuge for the maximum speed for the 30 min. Remove supernatant.
Wash Step
8. Add enough amount of ethanol to the pellet in order to wash
9. Then add 50 µl of TE – buffer
10. After that, add 0.5 µl of the RNAase
11. There is a need to incubate
12. Then incubate for the 37°C for 1 hour
13. Inactivate at 60°C for 10 min
14. Then run the gel
PUT PHOTO OF THE EXTRACTED GENOME
2.06.15
#1
Construction of the light system
FixK2 promoter(K592006) + rbs + tetR(C0040) + terminator + terminator + Ptet(R0040) + rbs + RFP(J06505) + terminator + terminator
[rbs] = 139.15 ng/µl
[Ptet + GFP] = 199.2 ng/µl
[pFixK2] = 131.6 ng/µl
[double terminator] = 70.21 ng/µl
[tetR] = 78.76 ng/µl
Restriction digest of pFixK2 and RBS
1. BBa_K592006 FixK2 promoter is the 250 base pairs long. It was cut with NEB enzymes, EcoRI and SpeI.
· pFixK2 – 7.6 µl
· EcoRI = 0.5 µl
· SpeI = 0.5 µl
· dH2O = 36.4 µl
· cut smart = 5 µl Overall: 50 µl
2. Ribosome binding site (15 base pairs) was cut with SpeI NEB Enzyme
· RBS = 7.2 µl
· XbaI = 1 µl
· Cut smart = 5 µl
· dH2O = 36.8 µl Overall: 50 µl
Restriction digest of tetR and double terminator
1. tetR (685 base pairs) was cut with EcoRI and SpeI.
· tetR = 12.7 µl
· EcoRI= 0.5 µl
· SpeI = 0.5 µl
· Cut smart = 5 µl
· dH2O = 31.3 µl Overall: 50 µl
2. Double terminator (95 base pairs )
· dTer = 14.2 µl
· XbaI = 1 µl
· Cut Smart = 5 µl
· dH2O = 29.8 µl Overall: 50 µl
Gel extraction of the pFixK2, RBS, tetR and double terminator
1. Invitrogen by life Technologies PureLink Quick Gel Extraction Kit was used to purify DNA.
2. The small area of the gel containing the DNA fragment of interest was cut under UV.
3. Mass of FixK2 = 220 mg
RBS = 80 mg
tetR = 150 mg
dTer = 140 mg
4. The protocol of the PureLink Quick Gel Extraction was used to dissolve the gel and extract the DNA
5. [FixK2] = 5.727 ng/µl
[Rbs] = 4.499 ng/µl
[tetR] = 5.216 ng/µl
[double terminator] = 2.694 ng/µl
Ligation of the Parts
pFixK2+ rbs
· pFixK2 = 8.5 µl
· RBS = 8.5 µl
· T4 ligase = 1 µl
· T4 buffer = 2 µl Overall: 20 µl
tetR +double terminator
· tetR = 8.5 µl
· double terminator= 8.5 µl
· T4 ligase = 1 µl
· T4 buffer = 2 µl Overall: 20 µl
Then ligated DNA was transformed and plate with pFixK2 with RBS grew on the plate.
The mini- prep of the pFixK2 + rbs was done
3.06.15
The concentration of the transformed ligated parts
FixK2+ rbs= 75.79 ng/µl
FixK2+ rbs= = 72.80 ng/µl
tetR+dter= 56. 93 ng/µl
tetR+dter= 95.86ng/µl
The concentration of the transformermed Pveg and FixJ
Pveg (BBa_K823003) and FixJ (BBa_K592016)
Pveg= 84.84 ng/µl
FixJ= 161.1 ng/µl
PCR (Thermo Scientific Phusion High Fidelity PCR Master- mix )
1. FixK2
· DNA = 0.03 µl *10 reactions = 0.3 µl
· Water = 5.97 µl= 59.7 µl
· Master Mix = 10 µl= 100 µl
· VF2 = 2µl = 20 µl
· VR= 2 µl = 20 µl
2. RBS
· DNA = 0.0277 µl = 0.287 µl
· Water= 5.9713 µl = 59.7 µl
· MM= 10 µl = 100 µl
· VF2= 2 µl = 20 µl
· VR=2 µl = 20 µl
3. FixK2+ rbs
· DNA= 0.06 =0.6
· Water= 5.94=59.4
· MM=10= 100
· VF2=2=20
· VR= 2=20
4. tetR+dter
· DNA= 0.07=0.7
· Water= 5.93=59.3
· MM= 10=100
· VF2= 2= 20
· VR=2 =20
5. tetR
· DNA= 0.05= 0.5
· Water= 5.95= 59.5
· MM= 10 =100
· VF2= 2= 20
· VR= 2= 20
6. Double terminator
· DNA= 0.06=0.6
· Water= 5.94= 59.4
· MM= 10 =100
· VF2=2= 20
· VR=2=20
Results; The ligation of the FixK2+ rbs did not work
The ligation of the tetR+ dter worked
The Restriction Digest of the tetR (BBa_C0040) and double terminator (BBa_K823017)
tetR
· Water= 31.3 µl
· DNA= 12.7
· EcoR1= 0.5
· SpeI= 0.5
· Cut Smart= 5
Double terminator
· Water= 29.8 µl
· DNA= 14.2
· EcoRI= 0.5
· XbaI = 0.5
· Cut Smart= 5 µl
Concentrations after Gel extraction
4.06.15
#1 PCR of the ligated parts after transformation and mini - prep
[FixK2+rbs+tetR] (950 base pairs) = 108.54 ng/µl
[tetR+ double terminator] = 166 ng/µl
1. FixK2+rbs+tetR
· High Fidelity Master Mix= 10*10 reactions= 100 µl
· DNA= 0.09=0.9 µl
· VF2 (0.5 µM) = 2 µl = 20 µl
· VR= 2 µl
· Sterile water =5.91 µl
2. tetR + double terminator
· High Fidelity Master Mix= 10µl = 100 µl
· tetR+ double terminator= 0.06= 0.6 µl
· VF2= 2 =20 µl
· VR= 2=20 µl
· Sterile water= 5.94= 59.4 µl
#2 Running on the gel of the parts after PCR
Order of the parts on the gel;
1. Ladder
2. FixK2+ RBS
3. FixK2+ rbs+tetR
4. tetR
5. tetR+ dter
5.06.15
Protocol for making the competent dh5alpha
1. Inoculate a single colony into 5 mL LB in 50 mL falcon tube (taped on a loosed tap)
2. Grow on 37°C with shaking for 130 rpm overnight
3. Use 1 mL to inoculate 100 mL to inoculate 100 mL of LB in 250 mL bottle the next morning
4. Shake 37°C for 1.5-3 hours
5. When OD is between 0.3-0.4 put cell on ICE
6. Hold cells on ice for the 10 minutes
7. Collect cells by centrifugation for 3 min at maximum speed (4700 rpm)
8. Decant supernatant and gently resuspend on 10 mL ice-cold 0.1 M CaCl2 (prepared in ddH2O). Treat them gently.
9. Incubate on ice for 20 minutes.
10. Centrifuge again at maximum speed (4700 rpm)
11. Discard supernatant and gently resuspend in 5 mL cold 0.1 M CaCl2 (15% glycerol)
12. Dispence into chilled microtubes, put on the dry ice. Perform this procedure very quickly!
13. Freeze in -80°C
6.06.15
#1
PCR clean-up of the [FixK2+rbs] = 30.15 ng/µl
[tetR] = 57.47 ng/
#2
[FixK2 + rbs] (PCR product digested) = 5.862 ng/µl
[tetR] = 4.621 ng/µl
#3
Fermentas Ligation tetR and double terminator
[tetR] = 20/4.621ng/µl = 4.3 µl
[FixK2+ rbs] = 40ng/5.862 ng/µl = 7 µl
Sterile water = 1µl
Ligase= 1 µl
Buffer = 2 µl
#4 Ligation of
· Pveg + FixJ
· FixK+ rbs+tetR
· tetR+double terminator
Ligation of tetR with double terminator with NEB- enzymes
1:1
50 ng: 50 ng
tetR = 7.518 ng/µl
tetR; 6.7 ng/ µl
double terminator; 3.3 µl
T4 ligase: 1 µl
T4 buffer: 2 µl
Sterile water: 7 µl
Pveg (19.59)+ FixJ (8.968)(Fermentas)
50ng: 50 ng
Pveg =2 µl
FixJ = 5.6 µl
T4= 1 µl
Buffer= 2 µl
Sterile water= 8.8 µl
FixK2+ rbs(10.66 ng/µl)+ tetR (23.08ng/µl) (NEB)
FixK2+ rbs = 4.7 µl
tetR= 2.2 µl
T4-ligase = 1µl
T4-buffer= 2 µl
Sterile water = 10.1 µl
tetR(4 ng/µl)+ double terminator (2ng/µl)(Fermentas)
tetR= 8 µl
double terminator= 9 µl
T4 ligase = 1 µl
Buffer= 2 µl
tetR + double terminator (Fermentas )
25 ng: 25 ng
tetR= 5 µl
double terminator=12 µl
T4 ligase = 1 µl
Buffer = 2 µl
Results; Ligation of the tetR + double terminator (NEB) 50 ng: 50 ng have worked
8.06.15
#1
Transformation of the Ligated products of the
· Pveg + FixJ
· tetR+ double terminator
· FixK2+rbs+tetR
· FixK2+rbs+tetR (PCR)
· FixK2+rbs+tetR
· FixK2+tetR+ double terminator
· tetR+ double terminator
9.06.15
#1
· Pveg +FixJ = 98.29 ng/µl
· tetR+ double terminator = 65.34 ng/µl
· FixK2+ rbs+ tetR = 83.32 ng/µl
· FixK2+ rbs+ tetR = 81.64 ng/µl
· tetR+ double terminator (NEB) = 101.3 ng/µl
· tetR+ double terminator = 100.4 ng/µl
#2
Polymerase chain Reaction
50µl
4
Master Mix
25µl
100 µl
tetR+ double terminator
0.15 µl
0.6 µl
VF2
5µl
20 µl
VR
5µl
20µl
H2O
14.8 µl
59.4 µl
50µl
4
Master Mix
25µl
100 µl
FixK2+rbs+ tetR
0.12 µl
0.48 µl
VF2
5µl
20µl
VR
5µl
20µl
H2O
14.88 µl
59.52 µl
50µl
4
Master Mix
25µl
100 µl
Pveg (237 base) + FixJ (1796)
0.1 µl
0.4 µl
VF2
5µl
20 µl
VR
5µl
20µl
H2O
14.9 µl
59.6 µl
50µl
4
Master Mix
25µl
100 µl
FixK2+rbs+tetR
0.12 µl
0.48 µl
VF2
5µl
20 µl
VR
5µl
20µl
H2O
14.88 µl
59.52µl
50µl
4
Master Mix
25µl
100 µl
tetR+ double terminator (Fermentas)
0.1 µl
59.6 µl
VF2
5µl
20 µl
VR
5µl
20µl
H2O
14.9 µl
59.4 µl
50µl
4
Master Mix
25µl
100 µl
tetR+ double terminator (fermentas) (2:1 fermentas)
0.1 µl
0.4 µl
VF2
5µl
20 µl
VR
5µl
20µl
H2O
14.9 µl
59.6 µl
Results: Only the ligation of the tetR + double terminator with NEB enzyme have worked
10.06.15
#1
Restriction Digest of the FixK2+ rbs with EcoRI and SpeI
DNA: 1000 ng/ 75.79ng/µl = 13.2 µl
EcoRI: 0.5 µl
SpeI: 0.5 µl
Cut smart: 5 µl
Sterile water: 30.8 µl
#2
Restriction Digest of tetR + double terminator with EcoRI and XbaI
DNA: 1000 ng/101.3 ng/µl = 9.87 µl
EcorI: 0.5 µl
XbaI: 0.5 µl
Cut smart: 5 µl
Sterile water: 34.13 µl
11.06.15
#1 Gel extract of the Pveg and FixJ
[Pveg ] = 3.174 ng/µl
[FixJ] = 2.9045 ng/µl
#2 Ligation of the Pveg and FixJ
T4 buffer: 2 µl
T4 ligase: 1 µl
Pveg: 25 ng/ 3.174ng/µl = 7.87= 8 µl
FixJ: 25 ng/2.9045 ng/µl = 8.61= 9 µl
12.06.15
№1 PCR of the S.mutans 16s rRNA with synthesized primers
S.mutans after gel extraction and genome isolation with Instagene Matrix
1 reaction
8 reactions
DNA
15µl
120 µl
Forward primer
5 µl
40 µl
Reverse primer
5 µl
40 µl
Master Mix
25 µl
200 µl
Sterile Water
0
0
TOTAL
50 µl
400 µl
Results:
№ 2 Single digest of the circular plasmid with EcoRI (NEB), SpeI (NEB), Aah1, and EcoRI (fermentas)
14.06.15
№1 S.mutans 16s rRNA PCR
1 reaction
8 reactions
DNA
15µl
120 µl
Forward primer
5 µl
40 µl
Reverse primer
5 µl
40 µl
Master Mix
25 µl
200 µl
Sterile Water
0
0
TOTAL
50 µl
400 µl
№2
Digest of the Pet 21 with NotI
· DNA= 1000/240.8 ng/µl = 4.15 µl
· NotI = 1µl
· Cut smart = 5 µl
· Sterile water = 39.85 µl
№3 Digest of the GFP with NotI
· DNA = 1000 ng/117.2 ng/µl = 8.53 µl
· NotI = 1µl
· Cut smart = 5 µl
· Sterile water = 35.47 µl
№4 Pveg and FixJ Ligation (2033 base pair)
№5 Digest of the FixK2+rbs with SpeI (NEB) (PCR product digest)
· DNA =1000ng/121.9 ng/µl = 8.2 µl
· SpeI = 1µl
· Cut smart = 5 µl
· Sterile water = 35.8 µl
№5 Digest of the FixK2+rbs with Aah1 (SibEnzyme) (PCR product digest)
· DNA = 1000 ng/121.9 ng/µl = 8.2 µl
· Aah1 = 1 µl
· SEB buffer = 5µl
· BSA= 0.5µl
· Sterile water = 35.3 µl
Results: The size of the FixK2+rbs (265 base pairs) do not coincide with the gel
15.06.15
№1 Solution on the day of the 15.06.2015
Transformation of the parts from Kit 2014
· Promoter FixK2 (Plate 1, 19G) – 250 base pair
· RBS (Plate 4, 4G) – 15 base pairs
· FixJ (Plate 1, 10N) – 1796 base pairs
· RFP mCherry with double terminator (Plate 1, 13K) = 861 b.p
· Promoter Veg (plate1, 20G) =
№2 Genome extraction from S.mutans with Instagene Matrix
№3
PCR of the 16S rRNA of the S.mutans
Photo will be here
16.06.2015
№ 1 Mini prep of of the parts
pFixK2 = 78.03 ng/µl
Rbs = 171.8 ng/µl
FixJ = 124.5 ng/µl
Rfp + double terminator = 82.75 ng/µl
Promoter Veg = 42.20 ng/µl
№ 2 Double Digest of the FixK2
DNA : 12.82 µl
Cut smart: 5 µl
SpeI:0.5 µl
EcoRI: 0.5 µl
Sterile water: 38.18 µl Overall: 50 µl
№3 Single digest of the FixK2
DNA: 12.82 µl
SpeI: 1 µl
Сut Smart Buffer: 5 µl
Sterile Water: 31.18 µl
№ 4 Double Digest of the RBS
DNA: 5.82 µl
EcoRI: 0.5 µl
XbaI : 0.5 µl
Buffer: 5 µl
Sterile water: 38. 18 µl
№ 5 Colony PCR
17.06.2015
№1 Gel extraction of the FixK2 and rbs
FixK2 (double digest) = 4.128 ng/µl
FixK2 (single digest) = 3.315 ng/µl
Rbs = 3.051 ng/µl
№2 Ligation of the FixK2 (double digest) + rbs
FixK2 = 7µl
Rbs = 9 µl
T4 ligase = 1 µl
T4 buffer = 2 µl
Sterile water = 1 µl
№ 3 Ligation of the FixK2(single digest )+rbs in order to obtain linear plasmid
FixK2 = 8 µl
rbs = 9 µl
T4 -ligase = 1 µl
T4 buffer = 2 µl
№ 4. Double digest of 1st line -Pveg (EcorI-SpeI), 2nd line FixJ (EcorI-XbaI), 3rd line RFP (EcorI - XbaI):
№ 5. Transformation of (Fixk2 + rbs) was done, one tube.
18. 06.2015
Double check Transformation of [Fixk2 + rbs]
We did not simultaneously perform digest and ligation since we wanted to check the work of the enzymes in double digest
2. Gel extraction of parts: Pveg, FixJ, RFP
3. Ligation reaction (rest parts):
Pveg + FixJ
Pveg +RFP. Reaction volumes for two reactions are the same:
ul = µl
Pveg = 24 ul
FixJ/RFP = 48 ul
Buffer = 8 ul
ligase = 4 ul
However, from now on we will use the the protocol DNA distribution according to 50 ng (insert) : 50 ng (backbone) notation
4. Chrm and Bacitracin plates were done (Bacitracin Mol. weight is 1422.71 g/mol and the concentration in stock (ex: 500 ml LB agar) should be 0.2 µM, while the conc. of bacitracin is 50 mg/ml):
(50 mg/ml)/ 1422. 71 = 0.035 M in eppendorf tube
0.035 M x Y= 0.2 µM x 500 ml
Y = 0.00286 ml = 2. 86 µl
Chrm conc: 500 µl for 500 ml of LB agar
5. MinElute Reaction cleanup kit was obtained (to purify after digestion and go directly to transformation) Ethanol (220 ml) was added
6. Kit has arrived!!!!!)
7. Miniprep of LB broth (Fixk2+rbs)
FixK2+ rbs = 217.7 ng/µl
19. 06. 2015
№ 1 Transformation of the ligated parts
1- agarplate ; dCas9 under xylose (2015 Kit, plate=5, 8L) chloromphenicol resistant
2- agar plate: Anderson promoter (plate 1, 20 K) chloromphenicol resistant
3-agar plate: Anderson promoter (plate1, 22A)
4-agar plate: Anderson promoter (plate 1, 22 K)
5-agar plate; GFP (plate 4, 21J)
6-agar plate; Mukhtars part (plate 5, 24D)
7-agar plate; P veg+ FixJ
8-agar plate; Pveg+ RFP
9-agar plate; Pveg+ FixJ (20 µl)
10 - agar plate: Pveg + RFP (20 µl )
2. PCR confirmation of the Fixk2+rbs ligation part obtained from miniprep
1- ladder, 2- FixK2, 3- FixK2+rbs, 4 - FixK2+ rbs
Photo will be here
Results; The size of the FixK2, FixK2 + rbs do not coincide with the right one.
3. Genome extraction from S.mutans by Instagene Genome Extraction and PCR
PCR
S.Mutans = 15 ul
P1 = 5 ul
P2 = 5 ul
MM (Buffer) = 25 ul
Fikx2 = 0.4 ul (on 3 tubes per 20 ul)
VF2 = 6 ul
VR = 6 ul
MM = 30 ul
water = 17.6 ul
RESULTS:
S.Mutans = No band amplification
FixK2 = 600 bp part was shown as amplified
Photo will be here!
20.06.2015
25.06.2015
№ 1 PCR of the S.mutans. Number 2 plate (grown from single colony). Genome isolated by Instagene Matrix
DNA = 15 ul
Primer Forward = 5 ul
Primer Reverse = 5 ul
Master Mix = 25 ul
№ 2 PCR of the S.mutans ( colony taken from Namber 2 plate)
DNA = 15 ul
Primer Forward = 5 ul
Primer Reverse = 5 ul
Master Mix = 25 ul
Results: The amplicon of the size 479 base pair is detected. First raw is the ladder, second is the amplicon which was PCR-ed with extension time 1 min, third is the amplicon with extension time with 14 seconds.
№3 Negative control with the Bacillus Subtilis genome. Genome of the Bacillus was extracted with Instagene matrix
26.06.2015
30.06.2015
№1 Transformation of the FixK2 ( Kit 2015, Plate 1, 19G)
№2 Restriction digest of the FixJ with the EcoRI and XbaI
DNA = 1000ng/124.5ng/ul = 8 ul
EcoRI = 1 ul
XbaI = 1 ul
Cut Smart = 5 ul
Sterile Water = 35 ul
Second raw after ladder is FixJ (already cut for gel extraction )
№3 Restriction Digest of the Pveg with EcoRI and SpeI
DNA = 1000 ng/42.20ng/ul = 24 ul
EcoRI = 1 ul
SpeI = 1 ul
Cut Smart = 5 ul
Sterile Water = 19 ul
№4 Gel extraction of the FixJ and Pveg
FixJ = 2.217 ng/ul
Pveg =2.892 ng/ul
№5 Ligation was performed in two ways.
DNA was taken from gel extraction
Pveg= 9 ul
FixJ = 8 ul
T4 -ligase = 1 ul
T4 -buffer = 2 ul
2. DNA was taken directly from digestion solution without clean up
Pveg = 8.5 ul
FixJ = 8.5 ul
T4 ligase= 1 ul
T4 buffer= 2 ul
07/07/15
Activity № 1
Results: Digest = sequential (1 line): 250 bp = Fixk2
PCR gradient: 4-11 line, 580 bp cmv promoter, actual size = 900 bp, digest of cmv was successful but , pcr shows that primers are impaired, since Master Mix and water were new and clean, dna was the same as for digest.
8.07.2015
Activity № 1. Sequential Digest of the Pveg with SpeI (sib enzyme) and EcoRI (fermentas)
Pveg = 12.5 ul
SpeI (sib) = 1 ul
BSA = 0.5 ul
Seb buffer = 5 ul
Sterile water = 31 ul
Incubation for 2 hours at 37 degree C
+ NaCl = 1 ul (5 M)
+BSA (0.5 ul)
+1 ul EcoRI (Fermentas)
Results: Pveg of the size 237 base pair was seen on the agarose gel
9.07.2015
Activity №1. Transformation
FixK2+rbs
Cmv+neomycin
GFP
Anderson promoter
Anderson promoter
Anderson promoter
Activity № 2. Digest of the RFP mcherry+ double terminator with Invitrogen EcoRI and XbaI
Results: The second line shows that there are two bands that are located very close to each other. This result suggests that upper band is the uncut plasmid, while the lower band is the one that was successfully digested. However, there is still doubt is it cut with one enzyme or both?
Activity № 3. Gel extract of the Pveg that was sequentially digested with SpeI (sib enzyme) and EcoRI (fermentas)
Pveg = 30.78 ng/ul
mcherryRFP + double terminator (digested with EX from Invitrogen) = 9.36 ng/ul
Activity № 4. Ligation of the Pveg+ mcherryRFP with double terminator
10x T4 DNA Ligase Buffer = 2 ul
T4-ligase = 1 ul
Pveg = 150 ng/30ng/ul = 4.87 ul
RFP = 50 ng/9.36 ng/ul = 5.34 ul
Sterile water = 6.79 ul
10.07.2015
Activity № 1. Checking of the Ligation of the Pveg+FixJ mini prep product by making sequential digest with SpeI (Sib enzyme) and EcoRI (fermentas)
Results: There are two bands. One is the 2500 bp. Another is 2000 base pair. However, if Pveg+ FixJ ligation have worked there would be also uncut plasmid after digestion of size 4500. So, it was decided to make single digest with EcorI in order to check if there would be a liniarized plasmid of 4500 base pair size.
Activity №2. Single digest of the Pveg+FixJ with the EcoRI (Neb enzyme)
DNA= 1000 ng/94.69 ng/ul = 10.56 ul
Cut smart = 5 ul
EcoRI = 1 ul
Sterile water = 33.44 ul
Overall: 50 ul
Results: First raw is ladder, second is plasmid after ligation of Pveg+FixJ, third is Pveg+FixJ plasmid cut with EcoRI. The results shows that there is no linearized plasmid with the 4500 base pair after ligation. So ligation did not work.
Activity №3. Transformation
Double Terminator =2014 Kit, plate 1, 3D, chloromphenicol
RBS = 2014 Kit, plate 4, 4G, chloromphanicol
Pveg= 2014 Kit, plate 1, 20G, chloromphenicol
Ligation product= Pveg + mcherry RFP (cut was done by EX of Invitrogen)