Team:Lethbridge/Notebook July

iGEM

9

PCR & Transformation

Thermo Scientific CloneJET PCR Cloning Kit (#K1232)

G-blocks to be ligated into pJET 1.2/blunt cloning vector

  1. RNAi 2015 iGEM TT ribozyme test construct (positive control)
  2. RNAi 2015 iGEM TT ribozyme test construct (GFP sense)
  3. RNAi 2015 iGEM TT ribozyme test construct (GFP anti-sense)
  1. Component Volume (μL)
    2x Reaction Buffer 10
    DNA fragment 1
    dH2O 6
    DNA blunting enzyme 1
    Total volume: 18μL
  2. Vortex briefly and centrifuge for 3-5 seconds
  3. Incubate the mixture at 70°C for 5 minutes and chill on ice
  4. Set up the ligation reaction on ice. Add the following to the blunting reaction mixture
    Component Volume (μL)
    pJET 1.2/blunt cloning vector (50ng/μL) 1
    T4 DNA ligase 1
    Total volume: 2μL
  5. Vortex briefly and centrifuge for 3-5 seconds
  6. Incubate the ligation mixture at room temperature (22°C) for 5 minutes. For PCR products >3kb, ligation can be prolonged to 30 minutes
  7. Use the ligation mixture directly for transformation. Keep the ligation mixture at -20°C if transformation is postponed. Thaw on ice and mix carefully before transformation

Note: Use only 1μL from this reaction mixture into 20μL of high efficiency cells

Transformation using High Efficiency Cells

  1. Thaw component DH5α cells
  2. Add 1μL of DNA to 25μL of competent DH5α cells
  3. Incubate on ice for 30 minutes
  4. Incubate at 42°C for 45 seconds
  5. Incubate on ice for 5 minutes
  6. Add 400μL of LB media
  7. Incubate while shaking (RNA common room 37°C shakers) for 1 hour
  8. Plate 300μL of cells on one plate
  9. Plate 100μL of cells on another plate

Note:

  • J23101 - pSBIC3
  • I13504 - pSBIA2
  • J23106 - pSBIC3
  • J23117 - pSBIC3
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13

Transformation

Transformation of BBA_I13504 using High Efficiency Cells

  1. Thaw component DH5α cells
  2. Add 1μL of DNA to 25μL of competent DH5α cells
  3. Incubate on ice for 30 minutes
  4. Incubate at 42°C for 45 seconds
  5. Incubate on ice for 5 minutes
  6. Add 400μL of LB media
  7. Incubate while shaking (RNA common room 37°C shakers) for 1 hour
  8. Plate 400μL of cells on one plate

3 devices to be built:

  • J23101 + I13504
  • J23106 + I13504
  • J23117 + I13504
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14

Miniprep & Glycerol Stock Protocol

Miniprepping Plasmid DNA (Bio Basic Inc)

  1. Add 2 mL overnight culture into a micro-centrifuge tube, centrifuge for 2 minutes at 12,000 rpm and pour off supernatant.
  2. Add 100μL of Solution I to the pellet, mix well and incubate at room temperature for 1 minute
  3. Add 200μL of Solution II to the pellet, mix by inverting and incubate at room temperature for 1 minute (do not vortex)
  4. Add 350μL of Solution III, mix by inverting and incubate at room temperature for 1 minute
  5. Centrifuge at 12,000 rpm for 5 minutes
  6. Transfer the supernatant to the EZ-10 column and centrifuge at 10,000 rpm for 2 minutes
  7. Discard the flow through, add 700μL of Wash Solution and centrifuge at 10,000 rpm for 2 minutes
  8. Repeat step 7
  9. Discard the flow through in the collection tube and spin at 10,000 rpm for 2 minutes (to remove residual EtOH)
  10. Transfer the column to a clean microcentrifuge tube. Add 50μL of elution buffer into the center of the column and incubate at room temperature for 2 minutes.
  11. Spin down sample for 2 minutes at 10,000 rpm and save flow through
  12. Store purified DNA at -20°C

Glycerol Stock Protocol

  1. Add 500μL of overnight culture to cryo-tube containing 500μL glycerol
  2. Pipette up and down to ensure homogeneity
  3. Place in liquid nitrogen to freeze
  4. Transfer to freezer box in -80°C freezer
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15

Single Digest & PCR

Single Digest of Miniprep DNA

  1. Set up digest reactions
    Component Volume (μL)
    dH2O 4.5
    10x Cut smart buffer (NEB) 1
    Miniprep DNA 4
    EcoRI (NEB) 0.5
    Total volume: 10μL
  2. Incubate at 37°C for 1 hour
  3. Run a 1% agarose gel on the single digest reactions
    Lane Component Volume (μL)
    1 1 kb DNA ladder 2μL
    2 J23106 DNA(1) + EcoRI 10μL of reaction + 2μL of dye
    3 J23117 DNA(1) + EcoRI 10μL of reaction + 2μL of dye
    4 J23117 DNA(2) + EcoRI 10μL of reaction + 2μL of dye
    5 I13504 DNA(1) + EcoRI 10μL of reaction + 2μL of dye
    6 I13504 DNA(2) + EcoRI 10μL of reaction + 2μL of dye
    7 J23103 DNA(1) + EcoRI 10μL of reaction + 2μL of dye
    8 TTr positive control DNA(1) + EcoRI 10μL of reaction + 2μL of dye
    9 TTr positive control DNA(2) + EcoRI 10μL of reaction + 2μL of dye
    10 TTr anti-sense DNA(1) + EcoRI 10μL of reaction + 2μL of dye
    11 TTr anti-sense DNA(2) + EcoRI 10μL of reaction + 2μL of dye
    12 TTr sense DNA(1) + EcoRI 10μL of reaction + 2μL of dye
    13 TTr sense DNA(2) + EcoRI 10μL of reaction + 2μL of dye
    14 1 kb DNA ladder 2μL
    Gel ran for 30 minutes at 135V

PCR of 3 TTr Pieces out of pJET

  1. Set up digest reactions
    Component Volume (μL)
    pJET forward primer (10μM) 0.5
    pJET reverse primer (10μM) 0.5
    Phusion DNA polymerase (10μM) 0.5
    DNA samples 1
    dNTPs 0.4
    Phusion HF buffer 5x 4
    MilliQ H2O 13.4
    Total volume: 20μL
  2. Incubate at 37°C for 1 hour
  3. Run the reactions under the following conditions
    1. Initial denaturation: 95°C for 5 minutes
    2. Repeat 35 times:
      1. 95°C for 20 seconds
      2. 72°C for 20 seconds
      3. 72°C for 45 seconds
    3. Final elongation: 72°C for 5 minutes
    4. Hold at 4°C
  4. Run an agarose gel using the PCR products
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16

Restriction Digest & Ligation

Restriction Digest of the Backbone DNA

  1. Set up double digests for the miniprep DNA
    1. Reaction for the J23106, J23101 and J23117 samples in the pSBIC 3 vector
      Component Volume (μL)
      10x CutSmart buffer 5
      Miniprep pDNA (47 - 561 ng/μL) 15
      MilliQ H2O 28
      SpeI (10U/μL) 1
      PstI (20U/μL) 1
      Total volume: 50μL
    2. Reaction for the I13504 in the pSBIA2 vector
      Component Volume (μL)
      10x CutSmart buffer 5
      Miniprep pDNA (47 - 561 ng/μL) 5
      MilliQ H2O 38
      XbaI (20U/μL) 1
      PstI (20U/μL) 1
      Total volume: 50μL
  2. Incubate at 37°C for 1 hour
  3. 1.00μL SAP to J23106, J23101 and J23117
  4. Column purify digests as well as gel extracted small GFP part

Ligation of I13504 Fragment into Different Backbones

Set up ligation reactions

  1. Reaction 1 (prepare 2, one to be incubated at 16°C and another at room temperature)
    Component Volume (μL)
    Fresh T4 DNA ligase buffer 10x 1
    T4 DNA ligase 0.5
    I13504 DNA part (gel extracted) 2
    J23101 backbone 1
    dH2O 5.5
    Total volume: 10μL
  2. Reaction 2
    Component Volume (μL)
    Fresh T4 DNA ligase buffer 10x 1
    T4 DNA ligase 0.5
    I13504 DNA part (gel extracted) 1.1
    J23117 backbone 0.2
    dH2O 7.5
    Total volume: 10μL
  3. Reaction 3
    Component Volume (μL)
    Fresh T4 DNA ligase buffer 10x 1
    T4 DNA ligase 0.5
    I13504 DNA part (gel extracted) 1
    J23106 backbone 1
    dH2O 5.5
    Total volume: 10μL
  4. Reaction 4
    Component Volume (μL)
    Fresh T4 DNA ligase buffer 10x 1
    T4 DNA ligase 0.5
    I13504 DNA part (gel extracted) 3
    J23101 backbone 0.5
    dH2O 5
    Total volume: 10μL
  5. Reaction 5
    Component Volume (μL)
    Fresh T4 DNA ligase buffer 10x 1
    T4 DNA ligase 0.5
    I13504 DNA part (gel extracted) 3
    J23117 backbone 0.2
    dH2O 5.3
    Total volume: 10μL
  6. Reaction 6
    Component Volume (μL)
    Fresh T4 DNA ligase buffer 10x 1
    T4 DNA ligase 0.5
    I13504 DNA part (gel extracted) 3
    J23106 backbone 0.5
    dH2O 5
    Total volume: 10μL
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17

PCR

PCR of the TT Ribozyme Test Constructs

  1. Set up PCR reactions
    1. Reaction using the TT ribozyme positive control DNA as template
      Component Volume (μL)
      pJET forward primer (10μM) 2
      pJET reverse primer (10μM) 2
      Pfu DNA polymerase (2.5U/μL) 1
      DNA template 0.75
      DNTPs (10mM) 4
      10x PCR reaction bufferx 10
      MilliQ H2O 80.25
      Total volume: 100μL
    2. Reactions using the TT ribozyme sense DNA and the TT ribozyme anti-sense DNA
      Component Volume (μL)
      pJET forward primer (10μM) 2
      pJET reverse primer (10μM) 2
      Pfu DNA polymerase (2.5U/μL) 1
      DNA template 0.5
      DNTPs (10mM) 4
      10x PCR reaction buffer 10
      MilliQ H2O 80.50
      Total volume: 100μL
  2. Run the reactions using the following cycle
    1. Initial denaturation: 95°C for 5 minutes
    2. Repeat 35 times:
      1. 95°C for 20 seconds
      2. 72°C for 20 seconds
      3. 72°C for 45 seconds
    3. Final elongation: 72°C for 5 minutes
    4. Hold at 4°C
  3. Run an agarose gel using the PCR products
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20

Miniprep, Agarose Gel & In Vitro Transcription

Miniprep Overnight Cultures

  • RNA affinity purification colonies 1-5 were picked, this part is in pJET1.2
  • Transformation #2 colonies 1-3
  • Transformation #3 colonies 1-3

Miniprepping Plasmid DNA (Bio Basic Inc)

  1. Add 2mL overnight culture into a micro-centrifuge tube, centrifuge for 2 minutes at 12,000 rpm and pour off supernatant.
  2. Add 100μL of Solution I to the pellet, mix well and incubate at room temperature for 1 minute
  3. Add 200μL of Solution II to the pellet, mix by inverting and incubate at room temperature for 1 minute (do not vortex)
  4. Add 350μL of Solution III, mix by inverting and incubate at room temperature for 1 minute
  5. Centrifuge at 12,000 rpm for 5 minutes
  6. Transfer the supernatant to the EZ-10 column and centrifuge at 10,000 rpm for 2 minutes
  7. Discard the flow through, add 700μL of Wash Solution and centrifuge at 10,000 rpm for 2 minutes
  8. Repeat step 7
  9. Discard the flow through in the collection tube and spin at 10,000 rpm for 2 minutes (to remove residual EtOH)
  10. Transfer the column to a clean microcentrifuge tube. Add 50 μL of elution buffer into the center of the column and incubate at room temperature for 2 minutes.
  11. Spin down sample for 2 minutes at 10,000 rpm and save flow through
  12. Store purified DNA at -20°C

Prepping DNA for Analysis on an Agarose Gel (11 Samples)

  1. Take 5μL of your miniprep and put it into a small PCR tube
  2. Add 3μL of dH2O
  3. Add 1μL of 10x cutsmart buffer (NEB)
  4. Add 1μL of EcoRI
  5. Digest for 1 hour at 37°C

Run a 2% Agarose Gel at 135V for 25 Minutes

Lane Contents
1 1kb ladder
2 Antisense PCR reaction
3 Sense PCR reaction
4 Positive control PCR reaction
5 RAP1
6 RAP2
7 RAP3
8 RAP4 glycerol stock
9 RAP5
10 Transformation 2A
11 Transformation 2B
12 Transformation 2C
13 Transformation 3A
14 Transformation 3B
15 Transformation 3C
16 1kb ladder

Lanes 2, 3 and 4: loaded with 2μL of PCR reaction + 8μL of dH2O + 2μL of 6x DNA loading dye. Miniprep samples: loaded with 10μL od digest + 6x DNA loading dye.

In Vitro Transcription

Performed depending on the quality of PCR samples

  1. Set up reactions
    Components Volume (μL)
    MilliQ H20 92.1
    5x transcription buffer (TraB) 60
    100 Mm DTT 30
    25 mM NTPs 36
    100 mM GMP 15
    0.5 U/μL iPPase 6
    T7-RNA-Polymerase 30
    40 U/μL RNase inhibitor 0.9
    PCR product 30
    Total volume: 300μL
  2. Incubate at 37°C ON and the digest with DNase I for 1 hour at 37°C
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21

Transformation

Retry Transformation of Ligation Reaction 1

  1. 2.88g urea
  2. 1.8 ml polyacrylamide
  3. 1.2 ml 5Xtbe
  4. Fill to 6ml dH2O
  5. Dissolve the urea
  6. Add 8μL TEMED
  7. 50μL APS

Transforming Ligations 1 and 4 - Gel Extraction vs Column purified ligations into DH5α cells.

Making 10 mL of 10x TT concentration = 50 mM. Add 0.09008 g of theophylline to 10 mL of dH2O

Reactions tested:

  • 10μL of final volume
  • 2x
  • 1μL RNA
  • 2μL 5x binding buffer
  • 1μL 50 mM TT
  • 2X
  • 1μL RNA
  • 1μL TAKM7 10x
  • 1μL 50 mM TT

Incubate ON + RT at 37°C

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22

Mass Spectroscopy

RAP OE I 1 hour of LB media under an IPTG inducible promoter. Added 5mL of ON culture to 500ml of culture at 11:20am

At 11:20 added 5ml of ON RAP culture and added 500μL of AMP

At OD = 0.6 add 500μL of IPTG and 500μL of AMP. Also take 1ml sample every hour.

Time Optical density (OD)
11:20 0.00
12:12 0.01
13:13 0.05
14:30 0.20
15:00 0.42
15:30 0.71
15:38 0.75
16:21 1.14
17:25 1.40
18:56 1.64
19:54 1.73
21:04 1.77
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27

Overexpression SDS PAGE

L1 PL
2 Time point 0
3 Time point 1
4 Time point 2
5 Time point 3
6 Time point 4
7 Time point 5
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28

PCR, Digests & Ligation

Digest pSBIC3 and Insert our MS2 Coding Sequence

Component Volume (μL)
pSCBIC3-RFP 10
Cutsmart buffer 10x 2
EcoRI 1
PstI 1
dH2O 7

Total volume: 20μL

  • 1 hour incubation
  • 1.0μL Rsap

PCR

Component Volume (μL)
10x PCR reaction buffer 10
DNA template RNAP3 0.5
10mM dNTPs 4
Forward primer → BB prefix 2
Reverse primer → BB suffix 2
MilliQ dH2O 80.5
Pfu DNA polymerase 1

Total volume: 100μL

PCR programme is pJET

Construct Digest

Component Volume (μL)
Construct 10
Cutsmart buffer 10x 2
EcoRI 1
PsfI 1
dH2O 7

Total volume: 20μL

1 hour incubation

Ligation Reaction

Component Volume (μL)
T4 DNA ligase buffer 1
T4 DNA ligase 0.5
pSBIC3 backbone 1
Construct 1
dH2O 6.5

Total volume: 10μL

Construct = 173.5μg/ml

Vector = 7.520μg/ml

Incubation overnight at room temperature

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29

Sequencing and Transformation

Sequencing

Sample Transformation Number
01 2A
02 2B
03 3A
04 3B
05 2A
06 2B
07 3A
08 3B

Transformation of ligation reactions 1 (at 16°C), 4 and construct + vector (L1)

Protocol:

  1. Add 1μL of DNA to 25μL of competent cells
  2. Incubate for 30 minutes on ice
  3. Heat shock at 47°C for 45 seconds
  4. Incubate on ice for 5 minutes
  5. Add 400μL of LB
  6. Incubate in RNA common room at 37°C for an hour
  7. Plate 400μL
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