Difference between revisions of "Team:NU Kazakhstan/Notebook"

Revision as of 06:04, 11 September 2015

Nazarbayev University Team

Notebook

1.06.15

1

Preparation of the LB agar We used the 37 g of nutrient agar for 400 mL of the distilled water

2

Extraction of genome from S.mutans 1. First, culture the S.mutans in 5 mL liquid BHI + bacitracin 2. Centrifuge at 15 min for 4000 rpm 3. 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, and Proteinase K. 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)

</html>

@@ Line 2: / Line 2: @@
 <html>
-<h2>Notebook</h2>
+<h2>Notebook</h2></html>
-<h3>MAKING COMPETENT CELLS</h3>
+.06.15
-<p>
-.	Inoculate a single colony into 5 mL LB in 50 mL falcon tube (taped on a loosed tap).</br>
+#1
-.	Grow on 37°C with shaking for 130 rpm overnight.</br>
+Preparation of the LB agar
-.	Use 1 mL to inoculate 100 mL to inoculate 100 mL of LB in 250 mL bottle the next morning.</br>
+We used the 37 g of nutrient agar for 400 mL of the distilled water
-.	Shake 37°C for 1.5-3 hours.</br>
+#2
-.	When OD is between 0.3-0.4 at 600 nm wavelength put cell on ice.</br>
+Extraction of genome from S.mutans
-.	Hold cells on ice for the 10 minutes.</br>
+.   First, culture the S.mutans in 5 mL liquid  BHI + bacitracin
-.	Collect cells by centrifugation for 3 min at maximum speed (4700 rpm).</br>
+.   Centrifuge at 15 min for 4000 rpm
-.	Decant supernatant and gently resuspend on 10 mL ice-cold 0.1 M CaCl2 (prepared in ddH2O). Treat them gently.</br>
+.   Then dissolve the pellet in 500 microliter of lysis buffer
-.	Incubate on ice for 20 minutes.</br>
-.	Centrifuge again at maximum speed (4700 rpm).</br>
+              	How to prepare Lysis Buffer (1 mL)
-.	Discard supernatant and gently resuspend in 5 mL cold 0.1 M CaCl2 (15% glycerol).</br>
+Lysis Buffer contains the EDTA, Tween 80%, tris HCl, and Proteinase K.
-.	Dispense into chilled microtubes, put on the dry ice. Perform this procedure very quickly.</br>
+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.
-.	Freeze in -80°C.</p>
+.   Incubation for the 2 hours at 55°C. Then there is a need to heat at the 90°C for 5 minutes.
-<h3>TRANSFORMATION</h3>
+.   Then add equal amount of cold isopropanol.
-<p>
+.   There is a need to incubate in freezer for the 20 minutes.
-.	Start thawing the competent cells on ice.</br>
+.   Centrifuge for the maximum speed for the 30 min. Remove supernatant.
-.	Add 50 µL of thawed competent cells into pre-chilled 2ml tube, and another 50µL into a 2ml tube, labelled for your control.</br>
+                         	Wash Step
-.	Add 1 - 2 µL of the resuspended DNA to the 2ml tube. Pipet up and down a few times, gently. Make sure to keep the competent cells on ice.</br>
+.   Add enough amount of ethanol to the pellet in order to wash
-.	Add 1 µL of the RFP Control to your control transformation.</br>
+.   Then add 50 µl of TE – buffer
-.	Close tubes and incubate the cells on ice for 30 minutes.</br>
+.               After that, add 0.5 µl of the RNAase
-.	Heat shock the cells by immersion in a pre-heated water bath at 42ºC for 60 seconds.</br>
+.               There is a need to incubate
-.	Incubate the cells on ice for 5 minutes.</br>
+.               Then incubate for the 37°C for 1 hour
-.	Add 200 μl of SOC media (make sure that the broth does not contain antibiotics and is not contaminated) to each transformation</br>
+.               Inactivate at 60°C for 10 min
-.	Incubate the cells at 37ºC for 2 hours while the tubes are rotating or shaking. Important: 2 hour recovery time helps in transformation efficiency, especially for plasmid backbones with antibiotic resistance other than ampicillin.</br>
+.               Then run the gel
-.	Label two petri dishes with LB agar and the appropriate antibiotic(s) with the part number, plasmid backbone, and antibiotic resistance. Plate 20 µl and 200 µl of the transformation onto the dishes, and spread. This helps ensure that you will be able to pick out a single colony.</br>
-.	For the control, label two petri dishes with LB agar (AMP). Plate 20 µl and 200 µl of the transformation onto the dishes, and spread.</br>
+              	PUT PHOTO OF THE EXTRACTED GENOME
-.	Incubate the plates at 37ºC for 12-14 hours, making sure the agar side of the plate is up. If incubated for too long the antibiotics start to break down and un-transformed cells will begin to grow. This is especially true for ampicillin - because the resistance enzyme is excreted by the bacteria, and inactivates the antibiotic outside of the bacteria.</br>
-.	You can pick a single colony, make a glycerol stock, grow up a cell culture and miniprep.</br>
-.	Count the colonies on the 20 μl control plate and calculate your competent cell efficiency.</p>
+.06.15
-<h3>LIGATION</h3>
-<p>
+#1
-.	Add 2ul of digested plasmid backbone (25 ng)</br>
+Construction of the light system
-.	Add equimolar amount of EcoRI-HF SpeI digested fragment (< 3 ul)</br>
+FixK2 promoter(K592006) + rbs + tetR(C0040) + terminator + terminator + Ptet(R0040) + rbs + RFP(J06505) + terminator + terminator
-.	Add equimolar amount of XbaI PstI digested fragment (< 3 ul)</br>
-.	Add 1 ul T4 DNA ligase buffer. Note: Do not use quick ligase</br>
+[rbs] = 139.15 ng/µl
-.	Add 0.5 ul T4 DNA ligase</br>
+[Ptet  + GFP] = 199.2 ng/µl
-.	Add water to 10 ul</br>
+[pFixK2] = 131.6 ng/µl
-.	Ligate 16C/30 min, heat kill 80C/20 min</br>
+[double terminator] = 70.21 ng/µl
-.	Transform with 1-2 ul of product</p>
+[tetR] = 78.76 ng/µl
-<h3>DNA EXTRACTION FROM CELLS (MINIPREP)</h3>
+                    	 Restriction digest of pFixK2 and RBS
-<p>
+.   BBa_K592006  FixK2 promoter is the 250 base pairs long. It was cut with NEB enzymes, EcoRI and SpeI.
-.	Harvest. Centrifuge 1-5 mL of the overnight LB-culture (Use 1-2×104 E.coli cells for each sample). Remove all medium. Add 2 mL of ddH2O and centrifuge again. Remove all medium.</br>
-.	Resuspend. Add 250 uL Resuspension Buffer (R3) with RNase A to the cell pellet and resuspend the pellet until it is homogenous.</br>
+·         pFixK2 – 7.6 µl
-.	Lyse. Add 250 uL Lysis Buffer (L7). Mix gently by inverting the capped tube until the mixture is homogenous. Do not vortex. Incubate the tube at room temperature for 5 minutes. </br>
+·         EcoRI  = 0.5 µl
-.	Precipitate. Add 350 uL Precipitation Buffer (N4). Mix immediately by inverting the tube, or for large pellets, vigorously shaking the tube, until the mixture is homogenous. Do not vortex. Centrifuge the lysate at >12,000 g for 10 minutes.</br>
+·         SpeI = 0.5 µl
-.	Bind. Load the supernatant from step 4 onto a spin column in a 2-mL wash tube. Centrifuge the column at 12,000 g for 1 minute. Discard the flow-through and place the column back into the wash tube.</br>
+·         dH2O = 36.4 µl
-.	Optional wash (Recommended for endA+ strains). Add 500 uL Wash Buffer (W10) with ethanol to the column. Incubate the column for 1 minute at room temperature. Centrifuge the column at 12,000 g for 1 minute. Discard the flow-through and place the column back into the wash tube.</br>
+·         cut smart = 5 µl   Overall: 50 µl
-.	Wash and remove ethanol. Add 700 uL Wash Buffer (W9) with ethanol to the column. Centrifuge the column at 12,000 g for 1 minute. Discard the flow-through and place the column back into the wash tube. Centrifuge the column at 12,000 g for 1 minute. Discard the flow-through.</br>
-.	Elute. Place the Spin Column in a clean 1.5-mL recovery tube. Add 75 uL of preheated TE Buffer (TE) to the center of the column. Incubate the column for 1 minute at room temperature.</br>
+.   Ribosome binding site (15 base pairs) was cut with SpeI NEB Enzyme
-.	Recover. Centrifuge the column at 12,000 g for 2 minutes. The recovery tube contains the purified plasmid DNA at 4⁰C (short-term) or store DNA in aliquots at -20⁰C (long-term).</p>
+·         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
+.   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
+.   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
+.   Invitrogen by life Technologies PureLink Quick Gel Extraction Kit was used to purify DNA.
+.   The small area of the gel containing the DNA fragment of interest was cut under UV.
+.   Mass of FixK2 = 220 mg
+        	RBS = 80 mg
+        	tetR = 150 mg
+        	dTer = 140 mg
+.   The protocol of the PureLink Quick Gel Extraction was used to dissolve the gel and extract the DNA
+.   [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
+.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 )
+.   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
+.   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
+.   FixK2+ rbs
+·         DNA= 0.06 =0.6
+·         Water= 5.94=59.4
+·         MM=10= 100
+·         VF2=2=20
+·         VR= 2=20
+.   tetR+dter
+·         DNA= 0.07=0.7
+·         Water= 5.93=59.3
+·         MM= 10=100
+·         VF2= 2= 20
+·         VR=2 =20
+.   tetR
+·         DNA= 0.05= 0.5
+·         Water= 5.95= 59.5
+·         MM= 10 =100
+·         VF2= 2= 20
+·         VR= 2= 20
+.   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
+.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
+.   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
+.   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;
+.   Ladder
+.   FixK2+ RBS
+.   FixK2+ rbs+tetR
+.   tetR
+.   tetR+ dter
+.06.15
+                         Protocol for making the competent dh5alpha
+.      Inoculate a single colony into 5 mL LB in 50 mL falcon tube (taped on a loosed tap)
+.      Grow on 37°C with shaking for 130 rpm overnight
+.      Use 1 mL to inoculate 100 mL to inoculate 100 mL of LB in 250 mL bottle the next morning
+.      Shake 37°C for 1.5-3 hours
+.      When OD is between 0.3-0.4 put cell on ICE
+.      Hold cells on ice for the 10 minutes
+.      Collect cells by centrifugation for 3 min at maximum speed (4700 rpm)
+.      Decant supernatant and gently resuspend on 10 mL ice-cold 0.1 M CaCl2  (prepared in ddH2O). Treat them gently.
+.      Incubate on ice for 20 minutes.
+.  Centrifuge again at maximum speed (4700 rpm)
+.  Discard supernatant and gently resuspend in 5 mL cold 0.1 M CaCl2 (15% glycerol)
+.  Dispence into chilled microtubes, put on the dry ice. Perform this procedure very quickly!
+.  Freeze in -80°C
+.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
+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)
+ng: 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 )
+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
+.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
+.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
+µl
+Master Mix
+µl
+µl
+tetR+ double terminator
+.15 µl
+.6 µl
+VF2
+µl
+µl
+VR
+µl
+µl
+H2O
+.8 µl
+.4 µl
+µl
+Master Mix
+µl
+µl
+FixK2+rbs+ tetR
+.12 µl
+.48 µl
+VF2
+µl
+µl
+VR
+µl
+µl
+H2O
+.88 µl
+.52 µl
+µl
+Master Mix
+µl
+µl
+Pveg (237 base) + FixJ (1796)
+.1 µl
+.4 µl
+VF2
+µl
+µl
+VR
+µl
+µl
+H2O
+.9 µl
+.6 µl
+µl
+Master Mix
+µl
+µl
+FixK2+rbs+tetR
+.12 µl
+.48 µl
+VF2
+µl
+µl
+VR
+µl
+µl
+H2O
+.88 µl
+.52µl
+µl
+Master Mix
+µl
+µl
+tetR+ double terminator (Fermentas)
+.1 µl
+.6 µl
+VF2
+µl
+µl
+VR
+µl
+µl
+H2O
+.9 µl
+.4 µl
+µl
+Master Mix
+µl
+µl
+tetR+ double terminator (fermentas) (2:1 fermentas)
+.1 µl
+.4 µl
+VF2
+µl
+µl
+VR
+µl
+µl
+H2O
+.9 µl
+.6 µl
+Results: Only the ligation of the tetR + double terminator with NEB enzyme have worked
+.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
+.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
+.06.15
+№1  PCR of the S.mutans 16s  rRNA with synthesized primers
+S.mutans after gel extraction and genome isolation with Instagene Matrix
+reaction
+reactions
+  	DNA
+µl
+µl
+ Forward primer
+µl
+µl
+Reverse primer
+µl
+µl
+Master Mix
+µl
+µl
+Sterile Water
+TOTAL
+µl
+µl
+Results:
+№ 2 Single digest of the circular plasmid with EcoRI (NEB), SpeI (NEB), Aah1, and EcoRI (fermentas)
+.06.15
+№1  S.mutans 16s rRNA PCR
+reaction
+reactions
+  	DNA
+µl
+µl
+ Forward primer
+µl
+µl
+Reverse primer
+µl
+µl
+Master Mix
+µl
+µl
+Sterile Water
+TOTAL
+µl
+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
+.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
+.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
+.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.
+. 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
+. Gel extraction of parts: Pveg,  FixJ, RFP
+. 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
+. 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
+.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
+. MinElute Reaction cleanup kit was obtained (to purify after digestion and go directly to transformation) Ethanol (220 ml) was added
+. Kit has arrived!!!!!)
+. Miniprep of LB broth (Fixk2+rbs)
+FixK2+ rbs = 217.7 ng/µl
+. 06. 2015
+№ 1 Transformation of the ligated parts
+- agarplate ; dCas9 under xylose  (2015 Kit, plate=5, 8L) chloromphenicol resistant
+- agar plate: Anderson promoter (plate 1, 20 K) chloromphenicol resistant
+-agar plate: Anderson promoter (plate1, 22A)
+-agar plate: Anderson promoter (plate 1, 22 K)
+-agar plate; GFP (plate 4, 21J)
+-agar plate; Mukhtars part (plate 5, 24D)
+-agar plate; P veg+ FixJ
+-agar plate; Pveg+ RFP
+-agar plate; Pveg+ FixJ  (20 µl)
+- agar plate: Pveg + RFP (20 µl )
+. PCR confirmation of the Fixk2+rbs ligation part obtained from miniprep
+- 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.
+. 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!
+.06.2015
+.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
+.06.2015
+.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
+. 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
+/07/15
+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.
+.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
+.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
+x 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
+.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)
-<h3>DNA EXTRACTION FROM GEL</h3>
-<h4>Excising and dissolving the gel</h4>
-<p>
-.	Equilibrate a water bath or heat block to 50⁰C.</br>
-.	Excise a minimal area of gel containing the DNA fragment of interest.</br>
-.	Weigh the gel slice containing the DNA fragment using a scale sensitive to 0.001 g.</br>
-.	Add Gel Solubilization Buffer (L3) to the excised gel in the tube size indicated in the following table:</br>
-Gel	Tube	Buffer L3 Volume</br>
-≤2% agarose	1.7-mL polypropylene	3:1 (i.e. 1.2 mL Buffer L3 : 400 mg gel piece)</br>
->2% agarose	5-mL polypropylene	6:1 (i.e. 2.4 mL Buffer L3 : 400 mg gel piece)</br>
-.	Place the tube with the gel slice and Buffer L3 into a 50⁰C water bath or heat block. Incubate the tube at 50⁰C for 10 minutes. Invert the tube every 3 minutes to mix and ensure gel dissolution.</br>
-.	After the gel slice appears dissolved, incubate the tube for an additional 5 minutes.</br>
-.	Optional: For optimal DNA yields, add 1 gel volume of isopropanol to the dissolved gel slice. Mix well.</br>
-.	Purify the DNA using a centrifuge.</p>
-<h4>Purifying DNA using a centrifuge</h4>
-<p>
-.	Load. Pipet the dissolved gel piece onto a Quick Gel Extraction Column inside a Wash Tube. Use 1 column per 400 mg of agarose gel. Note: the column reservoir capacity is 850 uL.</br>
-.	Bind. Centrifuge the column at >12,000 g for 1 minute. Discard the flow-through and place the column into the Wash Tube.</br>
-.	Wash. Add 50 uL Wash Buffer (W1) containing ethanol to the column.</br>
-.	Remove Buffer. Centrifuge the column at >12,000 g for 1 minute. Discard the flow-through and place the column into the Wash Tube. </br>
-Repeat Steps 3 and 4.</br>
-.	Remove Ethanol. Centrifuge the column at maximum speed for 3 minutes. Discard the flow-through.</br>
-.	Elute. Place the column into a Recovery Tube. Add 50 uL Elution Buffer (E5) to the center of the column. Incubate the tube for 2 minutes at room temperature.</br>
-.	Collect. Centrifuge the tube at >12,000 g for 5 minutes.</br>
-.	Store. The elution tube contains the purified DNA. Store the purified DNA at 4⁰C for immediate use or at -20⁰C for long-term storage.</p>
-<h3>DNA ISOLATION FROM BACTERIA</h3>
-<p>
-.	Pick an isolated bacterial colony and resuspend it in 1 mL of autoclaved water in a microfuge tube.</br>
-.	Centrifuge for 1 minute at 10,000-12,000 rpm. Remove the supernatant.</br>
-.	Add 200 uL of InstaGene matrix to the pellet and incubate at 56⁰c for 15-30 minutes.</br>
-Note: InstaGene matrix should be mixed at moderate speed on a magnetic stirrer to maintain the matrix in suspension. The pipet tip used should have a large bore, such as 1,000 uL pipet tip</br>
-.	Vortex at high speed for 10 seconds. Place the tube in a 100⁰C heat block or boiling water bath for 8 minutes.</br>
-.	Vortex at high speed for 10 seconds. Spin at 10,000-12,000 rpm for 2-3 minutes.</br>
-.	Use 20 uL of the resulting supernatant per 50 uL PCR reaction. Store the remainder of the supernatant at -20⁰C. Repeat Step 5 when reusing the InstaGene preparation.</br>
-Note: It is important to store the prepared sample at -20⁰C.
-</p>
-</div>
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