Team:NU Kazakhstan/Notebook

Nazarbayev University Team

Protocols

MAKING COMPETENT CELLS

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 at 600 nm wavelength 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. Dispense into chilled microtubes, put on the dry ice. Perform this procedure very quickly.
13. Freeze in -80°C.

TRANSFORMATION

1. Start thawing the competent cells on ice.
2. Add 50 µL of thawed competent cells into pre-chilled 2ml tube, and another 50µL into a 2ml tube, labelled for your control.
3. 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.
4. Add 1 µL of the RFP Control to your control transformation.
5. Close tubes and incubate the cells on ice for 30 minutes.
6. Heat shock the cells by immersion in a pre-heated water bath at 42ºC for 60 seconds.
7. Incubate the cells on ice for 5 minutes.
8. Add 200 μl of SOC media (make sure that the broth does not contain antibiotics and is not contaminated) to each transformation
9. 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.
10. 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.
11. 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.
12. 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.
13. You can pick a single colony, make a glycerol stock, grow up a cell culture and miniprep.
14. Count the colonies on the 20 μl control plate and calculate your competent cell efficiency.

LIGATION

1. Add 2ul of digested plasmid backbone (25 ng)
2. Add equimolar amount of EcoRI-HF SpeI digested fragment (< 3 ul)
3. Add equimolar amount of XbaI PstI digested fragment (< 3 ul)
4. Add 1 ul T4 DNA ligase buffer. Note: Do not use quick ligase
5. Add 0.5 ul T4 DNA ligase
6. Add water to 10 ul
7. Ligate 16C/30 min, heat kill 80C/20 min
8. Transform with 1-2 ul of product

DNA EXTRACTION FROM CELLS (MINIPREP)

1. 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.
2. Resuspend. Add 250 uL Resuspension Buffer (R3) with RNase A to the cell pellet and resuspend the pellet until it is homogenous.
3. 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.
4. 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.
5. 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.
6. 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.
7. 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.
8. 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.
9. 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).

DNA EXTRACTION FROM GEL

Excising and dissolving the gel

1. Equilibrate a water bath or heat block to 50⁰C.
2. Excise a minimal area of gel containing the DNA fragment of interest.
3. Weigh the gel slice containing the DNA fragment using a scale sensitive to 0.001 g.
4. Add Gel Solubilization Buffer (L3) to the excised gel in the tube size indicated in the following table:
Gel Tube Buffer L3 Volume
≤2% agarose 1.7-mL polypropylene 3:1 (i.e. 1.2 mL Buffer L3 : 400 mg gel piece)
>2% agarose 5-mL polypropylene 6:1 (i.e. 2.4 mL Buffer L3 : 400 mg gel piece)
5. 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.
6. After the gel slice appears dissolved, incubate the tube for an additional 5 minutes.
7. Optional: For optimal DNA yields, add 1 gel volume of isopropanol to the dissolved gel slice. Mix well.
8. Purify the DNA using a centrifuge.

Purifying DNA using a centrifuge

1. 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.
2. Bind. Centrifuge the column at >12,000 g for 1 minute. Discard the flow-through and place the column into the Wash Tube.
3. Wash. Add 50 uL Wash Buffer (W1) containing ethanol to the column.
4. Remove Buffer. Centrifuge the column at >12,000 g for 1 minute. Discard the flow-through and place the column into the Wash Tube.
Repeat Steps 3 and 4.
5. Remove Ethanol. Centrifuge the column at maximum speed for 3 minutes. Discard the flow-through.
6. 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.
7. Collect. Centrifuge the tube at >12,000 g for 5 minutes.
8. Store. The elution tube contains the purified DNA. Store the purified DNA at 4⁰C for immediate use or at -20⁰C for long-term storage.

DNA ISOLATION FROM BACTERIA

1. Pick an isolated bacterial colony and resuspend it in 1 mL of autoclaved water in a microfuge tube.
2. Centrifuge for 1 minute at 10,000-12,000 rpm. Remove the supernatant.
3. Add 200 uL of InstaGene matrix to the pellet and incubate at 56⁰c for 15-30 minutes.
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
4. Vortex at high speed for 10 seconds. Place the tube in a 100⁰C heat block or boiling water bath for 8 minutes.
5. Vortex at high speed for 10 seconds. Spin at 10,000-12,000 rpm for 2-3 minutes.
6. 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.
Note: It is important to store the prepared sample at -20⁰C.

Notebook

1.06.15

  1. Preparation of the LB agar
    We used 37 g of nutrient agar for 400 mL of distilled water
  2. Extraction of genome from S.mutans
    1. First, culture S.mutans in 5 mL liquid BHI + bacitracin
    2. Centrifuge for 15 min at 4000 rpm
    3. Then dissolve the pellet in 500 microliters of Lysis Buffer
      How to prepare Lysis Buffer (1 mL):
      Lysis Buffer contains EDTA, Tween 80%, tris HCl,125 microliters of 8 M EDTA,5 µl of Tween 80, Tris HCl 1M 50 µl, Proteinase K (200 µg/mL). 0.0002 grams of powder Proteinase K were put into 1 mL of Lysis Buffer. The balance could not read 0.0002 g of proteinase K, so 0.02 g of proteinase K were taken.
  3. Incubation for 2 hours at 55°C. Heat at 90°C for 5 minutes.
  4. Then add equal volume of cold isopropyl alcohol.
  5. Incubate in freezer for 20 minutes.
  6. Centrifuge at the maximum speed for 30 min. Remove the supernatant.
  7. Add enough amount of ethanol to the pellet in order to wash
  8. Then add 50 µl of TE buffer
  9. Add 0.5 µl of the RNAase
  10. Incubate at 37°C for 1 hour
  11. Inactivate at 60°C for 10 min
  12. Run it in an agarose gel

    13. 2.06.15

    1. Construction of the light system
      pFixK2(K592006) + rbs + tetR(C0040) + double terminator + Ptet(R0040) + rbs + RFP(J06505) + double 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
    2. Restriction digests:
      Protocol for Restriction digest:
      Take following amounts of reagents:
      -1000 ng of DNA
      -0.5 µl of each Restriction Enzyme(we used NEB enzymes)
      -5 µl of CutSmart Buffer
      -dH2O to get final volume of 50 µl
      Incubate this mixture at 37 C for 1-2 hours and heat inactivate for 20 min at the temperature needed for particular enzyme.
      Example mixture:
      -pFixK2 (250 bp) was cut with 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

      -RBS (15 bp) was cut with .
      -TetR (685 bp) was cut with EcoRI and SpeI.
      -Double terminator (95 bp) was cut with XbaI.
    3. 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
        • [FixK2] = 5.727 ng/µl
        • [Rbs] = 4.499 ng/µl
        • [tetR] = 5.216 ng/µl
        • [double terminator] = 2.694 ng/µl
      5. Ligation of the Parts:
        Protocol for Ligation:
        -Take 50 ng of vector or just of bigger DNA part
        -Take amount of smaller DNA part to have 1:3 molar ratio
        -1 µl of T4 ligase enzyme
        -2 µl of T4 buffer
        -Add dH2O to have final volume of 20 µl
        Incubate this mixture for 16 hours at 16 C and heat inactivate for 10 min at 65 C.
        Example Ligation mixture:
        -Ligation of pFixK2 + rbs
        • pFixK2 = 8.5 µl
        • RBS = 8.5 µl
        • T4 ligase = 1 µl
        • T4 buffer = 2 µl
        • Overall: 20 µl

        -Ligation of TetR + double terminator
        When ligated DNA was transformed, the plate with pFixK2 + RBS had colonies grown. The mini-prep of pFixK2 + rbs was done

        6. 3.06.15

        1. The concentrations of the transformed parts:
          • FixK2+ rbs= 75.79 ng/µl
          • FixK2+ rbs= = 72.80 ng/µl
          • TetR + dter= 56. 93 ng/µl
          • TetR + dter= 95.86ng/µl
          • Pveg= 84.84 ng/µl
          • FixJ= 161.1 ng/µl
        2. PCR (Thermo Scientific Phusion High Fidelity PCR Master-mix):
          Protocol for PCR reaction:
          Take following reagents:
          -10 ng of DNA
          -10 µl of PCR MasterMix
          -2 µl of each 5 µM Primer
          -Add dH2O to get final volume of 20 µl
          Example PCR mixture:
          -PCR of 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
              • -PCR of RBS
              -PCR of FixK2 + rbs
              -PCR of tetR+dter
              -PCR of tetR
              -PCR of Double terminator
              Result:
              -The ligation of the FixK2+ rbs did not work
              -The ligation of the tetR+ dter worked
            • Restriction Digests of:
              -tetR
              -Double terminator

        4.06.15

        1. PCR of the ligated parts after transformation and mini-prep:
          -PCR of [FixK2+rbs+tetR] = 108.54 ng/µl
          -PCR of [tetR+ double terminator] = 166 ng/µl
        2. Running of PCR products on a gel in the following order:
          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:
          -[FixK2+rbs] = 30.15 ng/µl
          -[tetR] = 57.47 ng/
        2. Gel extraction after digestion:
          -[FixK2 + rbs] = 5.862 ng/µl
          -[tetR] = 4.621 ng/µl
        3. Ligation with Fermentas enzymes:
          -[tetR] = 20/4.621ng/µl = 4.3 µl + [FixK2+ rbs] = 40ng/5.862 ng/µl = 7 µl
          -[tetR]=4 ng/µl + [double terminator]=2ng/µl
          -TetR + double terminator
          -Pveg + FixJ
        4. Ligation with NEB enzymes:
          -Pveg + FixJ
          -FixK+ rbs+tetR
          -[FixK2 + rbs]=10.66 ng/µl + [tetR]=23.08ng/µl
          -TetR+double terminator
        Results: Ligation of tetR + double terminator (NEB) 50 ng: 50 ng have worked.

        8.06.15

        Transformation of Ligated products:

        • 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

        PCR after ligation:
        -tetR+ double terminator
        -FixK2+rbs+ tetR
        -Pveg + FixJ
        -FixK2+rbs+tetR
        -tetR+ double terminator
        -tetR+ double terminator (fermentas)
        Results: Only ligation of the tetR + double terminator with NEB enzyme have worked

        10.06.15

        Restriction Digest:
        -FixK2+ rbs
        -TetR + double terminator

        11.06.15

        1.Gel extracts of:
        [Pveg ] = 3.174 ng/µl
        [FixJ] = 2.9045 ng/µl
        2.Ligation of Pveg + FixJ

        12.06.15

        PCR of S.mutans 16s rRNA with synthesized primers
        Figure 1: There were two types of colonies growing on bacitracin selective plate. They are expected to be S. mutans and S.sobrinus. It was suggested that Sm479F/R primer pair is highly specific for identification of S.mutans(Chen et al.,2007). In the picture you see two sets of lanes corresponding to the two types of colonies from plate, and first set of lanes is of correct size.

        14.06.15

        1.Digest of the Pet 21(plasmid) with NotI
        2.Digest of the GFP with NotI
        3.Ligation of Pveg with FixJ
        4.Digest of FixK2+rbs with SpeI (NEB)
        5.Digest of FixK2+rbs with Aah1 (Syberian Enzyme)
        Results: The size of FixK2+rbs (265 bp) does not coincide with the gel

        15.06.15

        1.Transformation of parts from Kit 2014:

        • Promoter FixK2 (Plate 1, 19G) – 250 bp
        • RBS (Plate 4, 4G) – 15 bp
        • FixJ (Plate 1, 10N) – 1796 bp
        • RFP mCherry with double terminator (Plate 1, 13K) = 861 bp
        • Promoter Veg (plate1, 20G) = 237 bp
        2.Genome extraction from S.mutans with Instagene Matrix
        3.PCR of 16S rRNA of the S.mutans

        16.06.2015

        1.Mini-prep:

        1. pFixK2 = 78.03 ng/µl
        2. Rbs = 171.8 ng/µl
        3. FixJ = 124.5 ng/µl
        4. Rfp + double terminator = 82.75 ng/µl
        5. Promoter Veg = 42.20 ng/µl
        2.Double Digest of FixK2 with SpeI and EcoRI
        3.Single digest of FixK2 with SpeI
        4.Double Digest of RBS with EcoRI and XbaI

        17.06.2015

        Gel extraction of 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 FixK2 (double digested) + rbs
        3.Ligation of FixK2(single digested) + rbs in order to obtain linear plasmid
        4.Double digest of:
        -Pveg (EcorI-SpeI)
        -FixJ (EcorI-XbaI)
        -RFP (EcorI - XbaI)
        5.Transformation of Fixk2 + rbs.

        18.06.2015

        1.Double check Transformation of [Fixk2 + rbs]
        We did not simultaneously perform digest and ligation since we wanted to check the work of enzymes in double digest
        2.Gel extraction of parts: Pveg, FixJ, RFP
        3.Ligation reaction:
        -Pveg + FixJ
        -Pveg +RFP
        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. Miniprep of Fixk2+rbs: [FixK2+ rbs] = 217.7 ng/µl

        19.06.2015

        1. Transformations of parts:
          1. dCas9 under xylose (2015 Kit, plate=5, 8L) chloromphenicol resistant
          2. Anderson promoter (plate 1, 20 K) chloromphenicol resistant
          3. Anderson promoter (plate1, 22A)
          4. Anderson promoter (plate 1, 22 K)
          5. GFP (plate 4, 21J)
          6. Pveg+ FixJ
          7. Pveg+ RFP
          8. Pveg+ FixJ (20 µl)
          9. 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
          Results: Size of FixK2, FixK2 + rbs do not coincide with the right one.
        3. Genome extraction from S.mutans by Instagene Genome Extraction and PCR
          RESULTS:
          S.Mutans = No band amplification
          FixK2 = 600 bp part was shown as amplified

        25.06.2015

        1. PCR of S.mutans. Plate #2 (grown from single colony). Genome isolated by Instagene Matrix
        2. PCR of S.mutans (colony taken from plate #2)
        3. Negative control with the Bacillus Subtilis genome. Genome of the Bacillus was extracted with Instagene matrix
        Results: The amplicon of 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 of 14 seconds.

        30.06.2015

        1. Transformation of FixK2 ( Kit 2015, Plate 1, 19G)
        2. Restriction digest of FixJ with EcoRI and XbaI
        3. Restriction Digest of Pveg with EcoRI and SpeI
        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
          -DNA was taken directly from digestion solution without clean-up

          6. 08.07.2015

          1. Sequential Digest of Pveg with SpeI (sib enzyme) and EcoRI (fermentas)
          Result: Pveg of size 237 bp was seen on an agarose gel

          9.07.2015

          1. Transformations:

          1. FixK2+rbs
          2. GFP
          3. Anderson promoter 101
          4. Anderson promoter 106
          5. Anderson promoter 117
            6. 2. Digest of the RFP mcherry + double terminator with Invitrogen EcoRI and XbaI
            3. Gel extraction of the Pveg that was sequentially digested with SpeI (syberian enzyme) and EcoRI (fermentas):
            Pveg = 30.78 ng/ul
            mcherryRFP + double terminator (digested with EX from Invitrogen) = 9.36 ng/ul
            4. Ligation of Pveg+ mcherryRFP with double terminator

            10.07.2015

            1. Checking Ligation of Pveg+FixJ mini prep product by making sequential digest with SpeI (Syberian enzyme) and EcoRI (fermentas)
            2. Single digest of Pveg+FixJ with the EcoRI (Neb enzyme)
            3. Transformation

            5.08.2015

            1.PCR of:

            • GFP
            • Anderson promoter 101
            • Anderson promoter 106
            • Anderson promoter 117
            • plasmid backbone
            • sgRNA for VicK

            6.08.2015

            1. CPEC of sgRNA for VicK and plasmid backbone.
            2. miniprep of pet21 plasmid from S.mutans
            3. restriction digest and ligation of GFP with each Anderson promoter

            7.08.2015

            1. PCR of ligation products of GFP with Anderson promoters

            8.08.2015

            1. CPEC of GFP+Anderson promoter part with plasmid backbone

            9.08.2015

            1. minipreps of pet21+sgRNA(Vick) and sgRNA(VicK) in a plasmid backbone
            2. PCR of pVeg and FixJ