Team:Lethbridge/Notebook July
PCR & Transformation
Thermo Scientific CloneJET PCR Cloning Kit (#K1232)
G-blocks to be ligated into pJET 1.2/blunt cloning vector
- RNAi 2015 iGEM TT ribozyme test construct (positive control)
- RNAi 2015 iGEM TT ribozyme test construct (GFP sense)
- RNAi 2015 iGEM TT ribozyme test construct (GFP anti-sense)
-
Component Volume (μL) 2x Reaction Buffer 10 DNA fragment 1 dH2O 6 DNA blunting enzyme 1 - Vortex briefly and centrifuge for 3-5 seconds
- Incubate the mixture at 70°C for 5 minutes and chill on ice
-
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 - Vortex briefly and centrifuge for 3-5 seconds
- Incubate the ligation mixture at room temperature (22°C) for 5 minutes. For PCR products >3kb, ligation can be prolonged to 30 minutes
- 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
- Thaw component DH5α cells
- Add 1μL of DNA to 25μL of competent DH5α cells
- Incubate on ice for 30 minutes
- Incubate at 42°C for 45 seconds
- Incubate on ice for 5 minutes
- Add 400μL of LB media
- Incubate while shaking (RNA common room 37°C shakers) for 1 hour
- Plate 300μL of cells on one plate
- Plate 100μL of cells on another plate
Note:
- J23101 - pSBIC3
- I13504 - pSBIA2
- J23106 - pSBIC3
- J23117 - pSBIC3
Transformation
Transformation of BBA_I13504 using High Efficiency Cells
- Thaw component DH5α cells
- Add 1μL of DNA to 25μL of competent DH5α cells
- Incubate on ice for 30 minutes
- Incubate at 42°C for 45 seconds
- Incubate on ice for 5 minutes
- Add 400μL of LB media
- Incubate while shaking (RNA common room 37°C shakers) for 1 hour
- Plate 400μL of cells on one plate
3 devices to be built:
- J23101 + I13504
- J23106 + I13504
- J23117 + I13504
Miniprep & Glycerol Stock Protocol
Miniprepping Plasmid DNA (Bio Basic Inc)
- Add 2 mL overnight culture into a micro-centrifuge tube, centrifuge for 2 minutes at 12,000 rpm and pour off supernatant.
- Add 100μL of Solution I to the pellet, mix well and incubate at room temperature for 1 minute
- Add 200μL of Solution II to the pellet, mix by inverting and incubate at room temperature for 1 minute (do not vortex)
- Add 350μL of Solution III, mix by inverting and incubate at room temperature for 1 minute
- Centrifuge at 12,000 rpm for 5 minutes
- Transfer the supernatant to the EZ-10 column and centrifuge at 10,000 rpm for 2 minutes
- Discard the flow through, add 700μL of Wash Solution and centrifuge at 10,000 rpm for 2 minutes
- Repeat step 7
- Discard the flow through in the collection tube and spin at 10,000 rpm for 2 minutes (to remove residual EtOH)
- 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.
- Spin down sample for 2 minutes at 10,000 rpm and save flow through
- Store purified DNA at -20°C
Glycerol Stock Protocol
- Add 500μL of overnight culture to cryo-tube containing 500μL glycerol
- Pipette up and down to ensure homogeneity
- Place in liquid nitrogen to freeze
- Transfer to freezer box in -80°C freezer
Single Digest & PCR
Single Digest of Miniprep DNA
- Set up digest reactions
Component Volume (μL) dH2O 4.5 10x Cut smart buffer (NEB) 1 Miniprep DNA 4 EcoRI (NEB) 0.5 - Incubate at 37°C for 1 hour
- 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
PCR of 3 TTr Pieces out of pJET
- 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 - Incubate at 37°C for 1 hour
- Run the reactions under the following conditions
- Initial denaturation: 95°C for 5 minutes
- Repeat 35 times:
- 95°C for 20 seconds
- 72°C for 20 seconds
- 72°C for 45 seconds
- Final elongation: 72°C for 5 minutes
- Hold at 4°C
- Run an agarose gel using the PCR products
Restriction Digest & Ligation
Restriction Digest of the Backbone DNA
- Set up double digests for the miniprep DNA
- 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 - 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
- Reaction for the J23106, J23101 and J23117 samples in the pSBIC 3 vector
- Incubate at 37°C for 1 hour
- 1.00μL SAP to J23106, J23101 and J23117
- Column purify digests as well as gel extracted small GFP part
Ligation of I13504 Fragment into Different Backbones
Set up ligation reactions
- 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 - 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 - 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 - 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 - 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 - 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
PCR
PCR of the TT Ribozyme Test Constructs
- Set up PCR reactions
- 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 - 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
- Reaction using the TT ribozyme positive control DNA as template
- Run the reactions using the following cycle
- Initial denaturation: 95°C for 5 minutes
- Repeat 35 times:
- 95°C for 20 seconds
- 72°C for 20 seconds
- 72°C for 45 seconds
- Final elongation: 72°C for 5 minutes
- Hold at 4°C
- Run an agarose gel using the PCR products
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)
- Add 2mL overnight culture into a micro-centrifuge tube, centrifuge for 2 minutes at 12,000 rpm and pour off supernatant.
- Add 100μL of Solution I to the pellet, mix well and incubate at room temperature for 1 minute
- Add 200μL of Solution II to the pellet, mix by inverting and incubate at room temperature for 1 minute (do not vortex)
- Add 350μL of Solution III, mix by inverting and incubate at room temperature for 1 minute
- Centrifuge at 12,000 rpm for 5 minutes
- Transfer the supernatant to the EZ-10 column and centrifuge at 10,000 rpm for 2 minutes
- Discard the flow through, add 700μL of Wash Solution and centrifuge at 10,000 rpm for 2 minutes
- Repeat step 7
- Discard the flow through in the collection tube and spin at 10,000 rpm for 2 minutes (to remove residual EtOH)
- 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.
- Spin down sample for 2 minutes at 10,000 rpm and save flow through
- Store purified DNA at -20°C
Prepping DNA for Analysis on an Agarose Gel (11 Samples)
- Take 5μL of your miniprep and put it into a small PCR tube
- Add 3μL of dH2O
- Add 1μL of 10x cutsmart buffer (NEB)
- Add 1μL of EcoRI
- 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
- 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 - Incubate at 37°C ON and the digest with DNase I for 1 hour at 37°C
Transformation
Retry Transformation of Ligation Reaction 1
- 2.88g urea
- 1.8 ml polyacrylamide
- 1.2 ml 5Xtbe
- Fill to 6ml dH2O
- Dissolve the urea
- Add 8μL TEMED
- 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
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 |
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 |
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
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:
- Add 1μL of DNA to 25μL of competent cells
- Incubate for 30 minutes on ice
- Heat shock at 47°C for 45 seconds
- Incubate on ice for 5 minutes
- Add 400μL of LB
- Incubate in RNA common room at 37°C for an hour
- Plate 400μL