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August
August 3, 2010
(in Lab: HB, AV, JV)
Objective: To restrict pBAD, sRBS, mRBS, TetR, dT and pTet for the assembly of pBAD-sRBS-TetR-dT-pTet
Method: Used Restriction of Plasmid DNA protocol.
- A front verctor was made made in sRBS ,mRBS, dT plasmids using EcoRI and Xbal enzymes
- pDNA that was cut out of plasmid for a front vector was pBAD, TetR, dT and pLacI using EcoRI and SpeI enzymes
- A back vector was made in sRBS and mRBS plasmids with PstI and SpeI
- pDNA that was cut out of plasmid for a back vector was TeTR and it was restricted with XbaI and PstI
Construct pDNA buffer Enzymes pBAD-sRBS/mRBS pBAD Red EcoRI and SpeI pBAD-sRBS/mRBS sRBS Orange XbaI and EcoRI pBAD-sRBS/mRBS mRBS Orange XbaI and EcoRII sRBS/mRBS-TetR sRBS Red PstI and SpeI sRBS/mRBS-TetR mRBS Red PstI and SpeI sRBS/mRBS-TetR TetR Tango XbaI and PstI TetR-dT TetR Red EcoRI and SpeI TetR-dT dT Orange XbaI and EcoRI dT-pTet dT Red EcoRI and SpeI dT-pTet pTet Orange XbaI and EcoRI pLAcI-sRBS/mRBS pLacI Red EcoRI and SpeI pLAcI-sRBS/mRBS sRBS Orange XbaI and EcoRI pLAcI-sRBS/mRBS mRBS Orange XbaI and EcoRI Mms6-PET28(a) PET28(a) Orange NotI
- For all reactions
- 158 (µL) Milli-Q H2O
- 10 (µL) Buffer
- 0.5(µL) of each enzyme
- 10 (µL) pDNA
Restriction was incubated for 1 hour at 37oC
Objective: Run PCR of pBAD, TetR, dT, pLacI, and mms6.
Method: Used PCR thermocycler iGEM program 7
Component Volume per tube (µL) Master Mix (x6) MilliQ H2O 41.8 250.8 10x Pfu Buffer with MgSO4 5 30 dNTPs 1 6 Forward Primer 0.5 3 Reverse Primer 0.5 3 Template DNA 1 - Pfu DNA polymerase 0.2 - Total 50 292.8
- Added 48.8 µL of Master Mix to each PCR reaction
Objective: Complete maxipreps of Lumazine (K249002), EYFP (E0030), and ECFP (E0020) and run on 1% agarose gel.
Method:
Lane Sample Components (µL) 1 1 kb ladder 2 loading dye (5x) + 0.5 ladder + 3.5 MilliQ H2O 2 ECFP 2 DNA + 2 loading dye (5x) + 2 MilliQ H2O 3 EYFP 2 DNA + 2 loading dye (5x) + 2 MilliQ H2O 4 Lumazine 2 DNA + 2 loading dye (5x) + 2 MilliQ H2O
- Ran at 100V for 42 minutes. Stained in EtBr for 15 minutes.
Results: AGAROSE GEL PICTURE
Objective: Ligate dT into pSB1C3.
Method:
- PCR amplify and purify both pSB1C3 and dT
- Restrict both with EcoRI and PstI
- Restrict pSB1C3 with DpnI
- Ligate pSB1C3 and dT
Restriction:
Restriction MilliQ H2O (µL) Buffer Orange (µL) pDNA (µL) Enzyme (µL) pSB1C3 79.25 10 10 0.25 EcoRI + 0.25 PstI + 0.25 DpnI pSB1C3 control 80 10 10 - dT 79.50 10 10 0.25 EcoRI + 0.25 PstI dT control 80 10 10 -
- Restriction were incubated at 37oC for 90 minutes.
- Enzymes were heat killed for 20 minutes at 80oC.
August 3, 2010 Evening
(in lab: KG, JS)
Objective: To run 1.5% agarose of restrictions: pBAD, sRBS, mRBS, TetR, dT and pTet for the assembly of pBAD-sRBS-TetR-dT-pTet
Method: Used a 1.5% agarose gel with 2 (µL) of loading dye and 10 (µL) of pDNA.
Lane Contents Result 1 1kb ladder 2 pTet (XbaI, EcoRI) good 3 pTet (orange buffer) --- 4 dT (XbaI, EcoR) no good 5 dT (orange buffer) --- 6 mRBS (SpeI, PstI) good 7 mRBS (red buffer) --- 8 sRBS (SpeI, PstI) good 9 sRBS (red buffer) --- 10 mRBS (XbaI, EcoRI) good 11 mRBS (orange buffer) --- 12 sRBS (XbaI, EcoRl) good 13 sRBS (orange buffer) --- 14 pet28(a) good 15 100 bp ladder 16 PSB1C3 not good 17 PSB1C3 restriction digest ---
Lane Contents Result 1 TetR (EcorI, SpeI) good 2 TetR (red buffer) --- 3 pLacI (EcoRI, SpeI) good 4 pLacI (red buffer) --- 5 Mms6 can not tell 6 Mms6 control --- 7 TetR (Xbal, PstI) good 8 TetR (tango buffer) --- 9 pBAD (EcoRI, SpeI) good 10 pBAD (red buffer) --- 11 dT (EcoRI, SpeI) good 12 dT (red buffer) --- 13 pLacI (2) ? 14 dT control not good 15 dT restriction 16 100 bp ladder 17 dT PCR product good 18 Mms6 PCR product good 19 pBAD PCR product good 20 pLacI PCR product good
Objective: To ligate: pBAD-sRBS/mRBS, sRBS/mRBS-TetR, TetR-dT, dT-pTet, pLacI-sRBS/mRBS, Mms6-ptet28(a), dT-PSB1C3
Method: Ligation of Plasmid DNA
15 (µL) pDNA in plasmid, and 15 (µL) of pDNA biobrick)
August 4, 2010
(in lab: JV)
Objective: PCR analysis of ligation product of August 3, 2010
- Ligations
- pBAD-mRBS
- pBAD-SRBS
- SRBS-tetR
- mRBS-TetR
- dt-pTet
- mms6-pET-28a
- dt-pSBIC3
- pLacI-SRBS
- Controls
- pBAD
- TetR
- TetR
- pLacI
- mms6
Method:
PCR: Thermocycler set to iGEM program 7
Component 1X(µL) Master Mix(x16)(µL) Milli-Q H2O 41.8 668.6 10x Pfu Buffer with MgSO4 5 80 dNTPs 1 16 Forward Primer 0.5 8 Reverse Primers 0.5 8 Template DNA 1 16 Pfu polymerase 0.2 3.2
2.5% agarose gel(1x TAE)
lane contents Successful Ligation ? 1 50bp ladder --- 2 dt pSBIC3 --- 3 dt pTet x 4 dt control --- 5 sRBS-TetR x 6 mRBS-TetR ? 7 TetR control --- 8 pLacI-mRBS x 9 pLacI-sRBS ? 10 pLacI control --- 11 Mms6 pET-28a no band 12 Mms6 control --- 13 pBad-SRBS x 14 pBad-mRBS x 15 pBad control ---
- Ran at 100V for 70 minutes.
Results: AGAROSE GEL PICTURE
Objective: Transform the successful ligations
Method: used Competent Cell Transformation protocol
- changes:
- used 50µL aliquottes of DH5&alpha
- did not pipette up and down once, the cells were just swirled 3 times
- added 400µL SOC media, shoock at 370C for 90 min
- platted 250µL and 150µL
Incubated from 12:00AM to 4;00 PM
results contents &250µL 150µL dt-pTet good x - control x x mms6-pET-28a good good dt-pSBIC3 x x mRBS-TetR good good pLacI-mRBS good good SRBS-TetR x x pBAD-SRBS good good + contol good good
August 6, 2010
In Lab: JV
Objective: Put lumazine synthase gene and mms6 into pET-28(A) for future overexpression.
Method: Restrict mms6 from Mr. Gene with NotI. Large quantities of DNA can be used so a gel extraction of the part can be done. PCR lumazine synthase out of its backbone. Restrict off the extra DNA fragments from the PCR with NotI. Restrict pET-28A with notI. Ligate.
Restriction:
Restriction MilliQ H2O (µL) Buffer Orange (µL) pDNA (µL) Enzyme (µL) mms6 799.5 100 100 1 NotI PCR:
Component 1X(µL) Milli-Q H2O 41.85 10x Pfu Buffer with MgSO4 5 dNTPs 1 Forward Primer (VF2) 0.5 Reverse Primers (VR) 0.5 Template DNA (Lumazine Synthase) 1 Pfu polymerase 0.2
2% Agarose Gel for Gel Extraction of mms6:
lane sample loaded 1 mms6 restricted with NotI 1 mL sample 2 mms6 unrestricted control 10(µL) sample, 2(µL)dye 3 50bp ladder 2(µL) ladder, 2(µL) dye, 8(µL) H20 3 1 kb ladder 2(µL) ladder, 2(µL) dye, 8(µL) H20
Gel ran for 60 minutes at 100 V. Small & faint band was slightly visible. Gel extraction was carried out using QIAGEN method. Eluted to 12 (µL).
August 6, 2010 Evening
Objective: Attempt colony pcr for rapid screening
Method: Followed two protocols from openwet
- Knight Protocol
- place 20(µL) sterile H2O in 0.6mL sterile tube
- with P10 pipette set to 3(µL) dip tip into colony
- place pipette tip into water and pipette up and down 20 times(this can be stored at 40C for inoculation of overnight 5mL cultures)
- Endy Protocol
- place 50(µL) sterile H2O in 0.6mL sterile tube
- with PLO pipette (set 3(µL)) dip sterile tip into colony
- place pipette tip into water and pipette up and down 20 times
Knight cont'd Endy cont'd setup 20(µL) reaction setup 20(µL) reaction 1(µL) colony suspension 2(µL) colony suspension 2(µL) 10x p.fu (+Mg SO4 2(µL) 10x p.fu (+Mg SO4 2(µL) dNTP 2(µL) dNTP 1.25(µL)VF2 Primer (10(µM) 0.75(µL)VF2 Primer (10(µM) 1.25(µL)VF Primer (10(µM) 0.75(µL)VF Primer (10(µM) 0.2(µL) Pfu polymerase 0.2(µL) Pfu polymerase 11.8 Milli-Q H2O 12.6 Milli-Q H2O
- as control for each rxn used equal volume of mRBS maxiprep
Knight cont'd Endy cont'd cycling conditions cycling conditions 950C for 15 minutes 950C for 6 minutes *940C for 30 seconds **950C for 30 seconds *560C for 30 seconds **560C for 30 seconds *680C for 1 minutes **700C for 1 minutes 680C for 20 minutes 700C for 10 minutes
(*) were run 39 times
(**) were run 35 times
Made the following program (called COLONYY) Lid preheat 980C
- 980C for 15 minutes
- 980C for 30 seconds
- 560C for 30 seconds
- 68-700C gradient for 1 minute
- 68-700C gradient for 20 minute
- 40C indefinte
bold selections were cycled 39 times
Objective: Analyzed PCR products on 2.5% TAE Agarose gel.
lane sample loaded 1 50bp ladder 1(µL) ladder, 1(µL) dye, 4(µL) H20 2 Knight control 5(µL) sample, 1(µL)dye 3 Knight colony 5(µL) sample, 1(µL)dye 4 Endy control 5(µL) sample, 1(µL)dye 5 Endy colony 5(µL) sample, 1(µL)dye 6 50bp ladder 1(µL) ladder, 1(µL) dye, 4(µL) H20 7 lumazine(justin's) 5(µL) sample, 1(µL)dye
Repeat gel with template controls
lane sample loaded 1 50bp ladder 0.5(µL) ladder, 2(µL) dye, 9.5(µL) H20 2 Endy Template 5(µL) colony suspension, 1(µL)dye, 4(µL) H20 3 Endy mRBS Control (PLR) 5(µL) sample, 1(µL)dye 4 Endy mRBS-TetR colony(PCR) 5(µL) sample, 1(µL)dye 4 mRBS template 0.5(µL) sample, 1(µL)dye, 4(µL) H20 5 Knight Template 0.25(µL) colony suspension, 1(µL)dye, 4(µL) H20 6 Knight mRBS control 5(µL) ladder, 1(µL) dye 7 Knight-mRBS-TetR colony 5(µL) sample, 1(µL)dye 1 1Kb ladder 0.5(µL) ladder, 2(µL) dye, 9.5(µL) H20
- ran at 100V for 75 minutes
Aug 9, 2010
(In Lab: HB)
Objective: Run PCR of mRBS-xylE I1 and I2 and lumazine.
Method:
PCR: Thermocycler set to iGEM program 7
Component 1X(µL) Master Mix(x4)(µL) Milli-Q H2O 41.8 167.2 10x Pfu Buffer with MgSO4 5 20 dNTPs 1 4 Forward Primer (VF2) 0.5 2 Reverse Primers (VR) 0.5 2 Template DNA 1 Pfu polymerase 0.2
Added 48.8µL Master Mix to each reaction tube.
Objective: Run 2% agarose gel to confirm PCR of mRBS-xylE I1 and I2 and lumazine worked.
lane sample loaded 1 50bp ladder 0.5(µL) ladder, 1(µL) dye, 6.5(µL) H20 2 mRBS-xylE I1 5(µL) sample, 2(µL)dye 3 mRBS-xylE I2 5(µL) sample, 2(µL)dye 4 Lumazine 5(µL) sample, 2(µL)dye
Ran at 100 V for 45 minutes. mRBS-xylE did not amplify while lumazine did amplify.
GEL PICTURE!
(In Lab: AV)
Objective: Prepared glycerol stocks & Miniprepped the following using the Qiagen Protocol.
pLacI-mRBS Colony 1 pLacI-mRBS Colony 2 pLacI-sRBS Colony 2 pLacI-sRBS Colony 3 pBAD-mRBS Colony 1 pBAD-mRBS Colony 2 pBAD-sRBS Colony 1 pBAD-sRBS Colony 2 dT-pTet Colony 1 dT-pTet Colony 3 mRBS-TetR Colony 1 mRBS-TetR Colony 3
Objective: To determine which of the previous ligations worked.
Method: Restricted with single cutter and double cutter.
Restriction Reaction (SINGLE)
Ingredient Volume(µL) MilliQ H20 Water 15.75 Orange Buffer (10x) 2 pDNA 2 EcoRI 0.25
Restriction Reaction (DOUBLE)
Ingredient Volume(µL) MilliQ H20 Water 15.50 Orange Buffer (10x) 2 pDNA 2 EcoRI 0.25 PstI 0.25
Unrestricted Control
Ingredient Volume(µL) MilliQ H20 Water 16 Orange Buffer (10x) 2 pDNA 2
DNA was restricted for 1 hour at 37oC.
Analyzed results on a 2% agarose gel with 2 (µL) of loading dye and 10 (µL) of pDNA. Load order as follows:
Lane Contents 1 1kb ladder 2 50 bp ladder 3 dT-pTet 1 DRD 4 dT-pTet 1 SRD 5 dT-pTet 1 URD 6 dT-pTet 3 DRD 7 dT-pTet 3 SRD 8 dT-pTet 3 URD 9 pLacI-mRBS 1 DRD 10 pLacI-mRBS 1 SRD 11 pLacI-mRBS 1 URD 12 pLacI-mRBS 2 DRD 13 pLacI-mRBS 2 SRD 14 pLacI-mRBS 2 URD 15 pLacI-sRBS 1 DRD 16 pLacI-sRBS 1 SRD 17 pLacI-sRBS 1 URD 18 pLacI-sRBS 3 DRD 19 pLacI-sRBS 3 SRD 20 pLacI-sRBS 3 URD
Lane Contents 1 1 kb ladder 2 50 bp ladder 3 pBAD-mRBS 1 DRD 4 pBAD-mRBS 1 SRD 5 pBAD-mRBS 1 URD 6 pBAD-mRBS 2 DRD 7 pBAD-mRBS 2 SRD 8 pBAD-mRBS 2 URD 9 pBAD-sRBS 1 DRD 10 pBAD-sRBS 1 SRD 11 pBAD-sRBS 1 URD 12 pBAD-sRBS 2DRD 13 pBAD-sRBS 2 SRD 14 pBAD-sRBS 2 URD 15 mRBS-TetR 1 DRD 16 mRBS-TetR 1 SRD 17 mRBS-TetR 1 URD 18 mRBS-TetR 3 DRD 19 mRBS-TetR 3 SRD 20 mRBS-TetR 3 URD
GEL PICTURE!
Aug 9,2010 Evening
(In Lab: JV, AS)
Objective: To ligate: lumazine into vector upstream of dT. Lumazine and mms6 into pET28a.
Method:
- Restrictions
Set up reactions as follows:
- Restrict Lumazine wit EcoRI and SpeI (Red Buffer)
- Restrict the dT with XbaI and EcoRI (Orange Buffer)
- Restrict Lumazine Synthase with NotI (Red Buffer)
Component Volume (µL) MilliQ H2O 15.6 or 15.8 Buffer 2 pDNA 2 Enzyme 0.20 + 0.20 Incubated reactions for 60 minutes at 37oC
- Ligation
Reaction set up as follows:Incubated reactions overnight at room temperature.
- T4 DNA ligase - 0.25µL
- DNA 1 - 8µL
- DNA 2 - 8µL
- 10x Ligation Buffer - 2µL
- MilliQ H2O - 1.75µL
Aug 10, 2010
(In Lab: JV)
Objective: Reran large gel from Aug 9/2010.
Load order was as follows:
Lane Contents 1 50 bp ladder 2 pBAD-mRBS 2 URD 3 pBAD-mRBS 2 SRD 4 pBAD-mRBS 2 DRD 5 pLacI-sRBS 3 URD 6 pLacI-sRBS 3 SRD 7 pLacI-sRBS 3 DRD 8 pLacI-mRBS 2 URD 9 pLacI-mRBS 2 SRD 10 pLacI-mRBS 1 DRD 11 dT-pTet 3 URD 12 dT-pTet 3 SRD 13 dT-pTet 3 DRD 14 pBAD-mRBS 1 URD 15 pBAD-mRBS 1 SRD 16 pBAD-mRBS 1 DRD 17 pLacI-sRBS 2 URD 18 pLacI-sRBS 2 SRD 19 pLacI-sRBS 2 DRD 20 1 kb ladder
Lane Contents 1 50 bp ladder 2 pLacI-mRBS 1 URD 3 pLacI-mRBS 1 SRD 4 pLacI-mRBS 1 DRD 5 dT-pTet 1 URD 6 dT-pTet 1 SRD 7 dT-pTet 1 DRD 8 mRBS-TetR 3 URD 9 mRBS-TetR 3 SRD 10 mRBS-TetR 3 DRD 11 mRBS-TetR 1 URD 12 mRBS-TetR 1 SRD 13 mRBS-TetR 1 DRD 14 pBAD-sRBS 2 URD 15 pBAD-sRBS 2 SRD 16 pBAD-sRBS 2 DRD 17 pBAD-sRBS 1 URD 18 pBAD-sRBS 1 SRD 19 pBAD-sRBS 1 DRD 20 1 kb ladder
GEL PICTURE!
(In Lab: JV)
Objective: Determine which ligations/transformations worked from 08/04/10.
Method: Colony PCR.
A: dT-pTet (1-10) B: pBAD-sRBS (1-10) C: pLacI-sRBS (1-10) D: pBAD-mRBS (1-10) E: mRBS-TetR (1-10) F: pLacI-mRBS (1-10)
Pick colony with pipette set at 3(µL) Pipette colony up and dow in 20(µL) sterile Milli-Q water. Will use 96 well plate.
PCR- Conditions: 1. 95oC for 15 min 2. 98oC for 20 sec 3. 55oC for 40 sec 4. 72oC for 2 min 5. 72oC for 20 min 6. 4oC infinitely
Reaction Mixture -
Method:
PCR: Thermocycler set to iGEM program 7
Component 1X(µL) Master Mix(x65)(µL) Milli-Q H2O 10.9 708.5 5X Phusion HF Buffer 4 260 dNTPs 1 65 Forward Primer (VF2) 1 65 Reverse Primers (VR) 1 65 Colony Template 2 Phusion polymerase 0.17 11.05
Controls: sRBS, pTET & mRBS - will PCR these to compare size using same conditions as above.
GEL PICTURE!
Aug 10, 2010 Evening
(In Lab: ADS)
Objective: PCR amplify xylE from mRBS-xylE for creation of xylE BioBrick
Method: 20µL reactions
PCR- Conditions: 1. Initial Denaturation 98oC for 30 sec 2. Denaturation 98oC for 10 sec 3. Anneal (51oC, 55oC,59.8oC, 64.6oC, 69.1oC, 71oC) for 30 sec 4. Extend 72oC for 30 sec 5. Final Extend 72oC for 10 min 6. Held 4oC for 30 hours
6 tubes in gradient PCR.
Component 1X(µL) Master Mix(x6.5)(µL) Milli-Q H2O 8.8 57.2 5X Phusion HF Buffer 4 26 dNTPs 2 13 Forward Primer 2 13 Reverse Primer 2 13 Template DNA 1 6.5 Polymerase 0.2 1.3
Ran samples on 1.5% agarose gel (1X TAE) for 60 minutes at 100V.
GEL PICTURE!
Aug 11, 2010
(In Lab: AS, JV, TF)
Objective: Determine what transformations have the correct insert from Aug. 4, 2010.
Method: Colony PCR. Changes - Used pipette tip instead of toothpick.
PCR: Thermocycler set to iGEM PFUTEST
1. pLacI-mRBS: 4 (A - E) 50(µL) 2. pBAD-mRBS: 5 (A - E) 20(µL) 3. mRBS-TetR: 6 (A -E) 20(µL) Controls - mRBS
Component 1X(µL) Master Mix(x7.5)(µL) Milli-Q H2O 9.8 73.5 10x Pfu Buffer with MgSO4 2 15 dNTPs 2 15 Forward Primer (VF2) 2 15 Reverse Primer (VR) 2 15 Template DNA 2 Pfu polymerase 0.2 1.5
Added 18µL Master Mix to each reaction tube.
(In Lab: ADS)
Objective: Transform mRBS-xylE BioBrick into DH5alpha.
Transformation -
A) Thawed 50(µL) Sub-Cloning Efficiency DH5alpha Competent Cells on ice. B) Gently mixed cells and then aliquoted 100(µL) into chilled polypropylene tubes. C) Added 2(µL) of BioBrick to cells. Added 5(µL) of pUC19 DNA to 100(µL) cells to determine efficiency. D) Incubated cells on ice for 30 minutes. E) Heat shocked cells for 45 seconds in a 42oC water bath. F) Placed on ice for 5 minutes. G) Added 0.4 mL of room temperature SOC medium. H) Shook at 225 rpm for 1 hour. I) Diluted control cells 1:100 with SOC medium. J) Spread 100(µL) of this dilution on LB-Amp agar plates K) Spread 50 and 250(µL) of experimental cells on LB-Cam agar plates. L) Incubated overnight at 37oC
Aug 12, 2010
(In Lab: JV)
Objective: Determine if Adam's colony PCRs' from Aug. 11, 2010 worked.
Method: Samples were run on a 2.5% agarose gel (1X TAE) for 1 hour at 100V.
GEL PICTURE!
Results: Lanes 2, 3, 4 and 8 showed PCR amplification. Colonies chosen don't show the correct insert size.
(In Lab: JV)
Objective: Screen for colonies with the correct insert from Aug. 4, 2010 transformations.
Method: Colony PCR. Changes - Used pipette tip instead of toothpick. Put colony in 20(µL) autoclaved Milli-Q water.
PCR: Thermocycler set to iGEM PFUTEST
1. pLacI-sRBS: A (11 - 17) 2. pBAD-sRBS: B (11 - 17) 3. dT-pTet: C (11 - 17) 4. pLacI-mRBS: D (11-17) 5. pBAD-mRBS: E (11-17) 6. mRBS-TetR: F (11-17)
Controls - mRBS, pTet, sRBS
Component 1X(µL) Master Mix(x47)(µL) Milli-Q H2O 9.8 460.6 10x Pfu Buffer with MgSO4 2 94 dNTPs 2 94 Forward Primer (VF2) 2 94 Reverse Primer (VR) 2 94 Template DNA (Cell Lysate) 2 94 Pfu polymerase 0.2 9.4
Added 18µL Master Mix to each reaction tube. Analyzed PCR products on 2.5% TAE gel. GEL PICTURE!!!
(In Lab: ADS, KG)
Objective: Perform PCR on lumazine, mms6, xylE plasmids with prefix and suffix primers (these will tell us exact size without subtracting VF2/VR regions). If right will ligate into pET28a plasmids.
Method: Plasmids used included: 6 mms6 maxipreps. 4 lumazine maxipreps. 5 xylE maxipreps. 1 mRBS maxiprep
PCR: Thermocycler set to iGEM Program #11 PFU - P/S
PCR- Conditions: 1. Initial Denaturation 95oC for 3 min 2. Denaturation 95oC for 30 sec 3. Anneal (54oC) for 30 sec 4. Extend 72oC for 3 min 5. Final Extend 72oC for 15 min 6. Held 4oC infinitely (25 cycles)
Component 1X(µL) Master Mix(x16.5)(µL) Milli-Q H2O 9.8 161.7 10x Pfu Buffer with MgSO4 2 33 dNTPs 2 33 Forward Primer (Prefix) 2 33 Reverse Primer (Suffix Antisense) 2 33 Template DNA (Cell Lysate) 2 Pfu polymerase 0.2 3.3
Added 18µL Master Mix to each reaction tube. Analyzed PCR products on 2.5% TAE gel run at 100 V for 35 minutes. GEL PICTURE!!!
Aug 13, 2010
(In Lab: AS)
Objective: PCR amplify minipreps prepared on Aug 9/2010 to screen for properly assembled BioBricks.
Component 1X(µL) Master Mix(x12.5)(µL) Milli-Q H2O 41.85 523.1 10x Pfu Buffer with MgSO4 5 62.5 dNTPs 1 12.5 Forward Primer (VF2) 0.5 6.25 Reverse Primer (VR) 0.5 6.25 Template DNA 1 Pfu polymerase 0.15 1.888
Added 49µL Master Mix to each reaction tube.
(In Lab: AS)
Objective: PCR amplify pSB1A3, pSB1T3 and pSB1C3 for use in future 3 part assembly and subsequent growth for glycerol stock.
Component 1X(µL) Master Mix(x13.5)(µL) Milli-Q H2O 14.9 52.15 10x Pfu Buffer with MgSO4 2 7 dNTPs 1 3.5 Forward Primer (SB-prep-2) 0.7 2.45 Reverse Primer (SB-prep-3p) 0.7 2.45 Template DNA 0.5 Pfu polymerase 0.2 0.7
PCR- Conditions: 1. 94oC for 30 sec 2. 94oC for 30 sec 3. 55oC for 30 sec 4. 68oC for 3 min 5. 68oC for 10 min 6. 4oC infinitely (36 cycles)
Added 19.5µL Master Mix to each reaction tube. Analyzed products on 1% TAE agarose gel which ran for 60 minutes at 100 V.
GEL PICTURE!
Aug 14, 2010
(In Lab: AS)
2.5% agarose gel(1x TAE)
lane contents 1 pBAD-mRBS 1 2 pBAD-mRBS 2 3 pBAD-sRBS 1 4 pBAD-sRBS 2 5 mRBS-TetR 1 6 mRBS-TetR 3 7 50 bp Ladder 8 dT-pTet 1 9 dT-pTet 3 10 pLacI-mRBS 1 11 pLacI-mRBS 2 12 pLacI-sRBS 2 13 pLacI-sRBS 3 14 MT 15 MT 16 MT 17 MT 18 MT 19 MT 20 MT 21 No Lanes 22 No Lanes 23 No Lanes 24 No Lanes 25 No Lanes 26 No Lanes 27 No Lanes
lane contents 1 K249001 2 K249004 3 K249005 4 K249006 5 MT 6 K249008 7 K249008 (Qiagen) 8 K249014 9 K249017 10 50 bp Ladder 11 1 12 2 13 3 14 4 15 5 16 xylE-dT 17 Lumazine-dT 18 pLacI-sRBS 19 MT 20 MT 21 MT 22 MT 23 MT 24 MT 25 MT 26 MT 27 MT GEL PICTURE!
(In Lab: AS)
Objective: Insert mms6 and lumazine into pET28a using NotI restriction site.
Method: 1. Reserve 1(µL) of dirty PCR product for analysis. 2. Clean up PCR products using Qiagen prep. 3. Restrict 4 mms6 maxipreps and 4 lumazine maxipreps.
Restriction Reactions:
For lumazine and mms6 -
Ingredient 1X(µL) Master Mix(x8.5)(µL) MilliQ H20 Water 7.8 66.30 Orange Buffer (10x) 2 17 pDNA 10 NotI 0.2 1.7 Added 10 (µL) to each tube.
For pET28a -
Ingredient Reaction Mix(µL) MilliQ H20 Water 4.8 Orange Buffer (10x) 5 pDNA 40 NotI 0.2 Incubated at 37oC. Analyzed results on 2.5% TAE agarose gel which ran at 100 V for 50 minutes. GEL PICTURE!!!
Results: Lost all the DNA in the column clean-up step and will have to re-do.
Aug 14, 2010 Evening
(In Lab: AS)
Objective: Assemble mms6-dT and lumazine-dT using three antibiotic assembly.
Method: 1. PCR amplify BioBricks (Prefix/Suffix) 2. Restrict BioBricks 3. Ligate BioBricks into psB1C3 4. Confirm ligation by PCR analysis (VF2/VR) 5. Transform ligation mixes 6. Screen colonies with Colony PCR
PCR: Thermocycler set to iGEM program 11
Component 1X(µL) Master Mix(x10.5)(µL) Milli-Q H2O 10.8 113.4 10x Pfu Buffer with MgSO4 2 21 dNTPs 2 21 Forward Primer (Prefix) 2 21 Reverse Primers (Suffix Antisense) 2 21 Template DNA 1 Pfu polymerase 0.2 2.1
Added 19 (µL) to each tube.
Restriction Reactions:
For lumazine and mms6 -
Ingredient 1X(µL) Master Mix(x5.5)(µL) MilliQ H20 Water 11.6 63.8 Red Buffer (10x) 2 11 pDNA 6 EcoRI 0.2 1.1 SpeI 0.2 1.1 Added 14 (µL) to each tube.
For dT -
Ingredient Reaction Mix(µL) MilliQ H20 Water 58 Tango Buffer (10x) 10 pDNA 30 XbaI 1 PstI 1 For psB1C3 -
Ingredient Reaction Mix(µL) MilliQ H20 Water 70 Orange Buffer (10x) 10 pDNA 30 EcoRI 1 PstI 1 DpnI 1 Also cut lumazine, mms6 and dT with one enzyme for two part, PCR amplification and subsequent ligation into pSB1X3.
For lumazine and mms6, CUT with SpeI -
Ingredient 1X(µL) Master Mix(x5.5)(µL) MilliQ H20 Water 15.8 86.9 Tango Buffer (10x) 2 11 pDNA 2 SpeI 0.2 1.1 Added 18 (µL) to each reaction.
For dT, CUT with XbaI-
Ingredient Reaction Mix(µL) MilliQ H20 Water 79 Tango Buffer (10x) 10 pDNA 10 XbaI 1 Incubated at 37oC for 1.5 hours. Heat killed enzymes for 20 minutes at 80oC.
Ligation Reactions:
3 Part: Lumazine/mms6 + dT + psB1C3
Ingredient 1X(µL) Master Mix(x5.5)(µL) MilliQ H20 Water 11.0 64.9 T4 Ligase Buffer (10x) 2 11 Plasmid (psB1C3) 2 11 Part 1 (Lumazine/mms6) 2 Part 2 (dT) 2 11 T4 DNA Ligase 0.2 1.1 2 Part: Lumazine/mms6 + dT
Ingredient 1X(µL) Master Mix(x5.5)(µL) MilliQ H20 Water 13.8 75.9 T4 Ligase Buffer (10x) 2 11 Part 1 (Lumazine/mms6) 2 Part 2 (dT) 2 11 T4 DNA Ligase 0.2 1.1 Added 18(µL) to each rxn tube. Incubated 1 hour and overnight at room temperature ( 25oC).
Screening via PCR amplification : Thermocycler set to iGEM program 11
3 Part: Lum/mms6 + dT + pSB1C3
Component 1X(µL) Master Mix(x5.5)(µL) Milli-Q H2O 33.8 185.9 10x Pfu Buffer with MgSO4 5 27.5 dNTPs 2 11 VF2 Primer 2 11 VR Primer 2 11 Template DNA 5 Pfu polymerase 0.2 1.1
Added 45 (µL) MM to each tube.
2 Part: Lum/mms6 + dT
Component 1X(µL) Master Mix(x5.5)(µL) Milli-Q H2O 6.8 37.4 10x Pfu Buffer with MgSO4 2 11 dNTPs 2 11 Prefix Primer 2 11 Suffix Antisense Primer 2 11 Template DNA 5 Pfu polymerase 0.2 1.1
Added 15 (µL) MM to each tube.
Aug 15, 2010
(In Lab: ADS)
Objective: Insert mms6 and lumazine into pET28a using NotI restriction site.
Method: 1. Reserve 1(µL) of dirty PCR product for analysis. 2. Clean up PCR products using Qiagen prep. 3. Restrict 3 mms6 maxipreps and 2 lumazine maxipreps.
Restriction Reactions:
For lumazine and mms6 -
Ingredient 1X(µL) Master Mix(x10.5)(µL) MilliQ H20 Water 11.8 123.9 Orange Buffer (10x) 2 21 pDNA 6 NotI 0.2 2.1 Added 14 (µL) to each tube.
For pET28a -
Ingredient Reaction Mix(µL) MilliQ H20 Water 4.8 Orange Buffer (10x) 5 pDNA 40 NotI 0.2 Incubated at 37oC for 1.5 hours. Heat killed enzymes at 80oC for 20 minutes.
Ligation Reactions:
Ingredient 1X(µL) Master Mix(x5.5)(µL) MilliQ H20 Water 13.9 75.9 T4 Ligase Buffer (10x) 2 11 Plasmid (pET28a) 2 11 Cut out part (Lumazine/mms6) 2 T4 DNA Ligase 0.2 1.1 Added 18(µL) to each tube. Incubated 1 hour and overnight at room temperature.
Aug 15, 2010 Evening
(In Lab: AS)
Objective: Assemble Lum-dT & mms6-dT using BioBrick standard assembly.
Method: Obtain plasmid DNA from maxipreps.
Restriction Reactions:
For lumazine and mms6 -
Ingredient 1X(µL) Master Mix(x5.5)(µL) MilliQ H20 Water 7.6 41.8 Red Buffer (10x) 2 11 pDNA 10 EcoRI 0.2 1.1 SpeI 0.2 1.1 For dT -
Ingredient Reaction Mix(µL) MilliQ H20 Water 38 Orange Buffer (10x) 10 pDNA 50 XbaI 1 EcoRI 1 Incubated at 37oC for 1.5 hours. Heat killed enzymes for 20 minutes at 80oC.
Ligation Reactions:
Ingredient 1X(µL) Master Mix(x5.5)(µL) MilliQ H20 Water 13.8 75.9 T4 Ligase Buffer (10x) 2 11 Plasmid (dT) 2 11 Cut out part (Lumazine/mms6) 2 T4 DNA Ligase 0.2 1.1 Added 18(µL) MM to each rxn tube. Incubated at one hour and overnight at room temperature.
Screening via PCR amplification : Thermocycler set to iGEM program 11
Component 1X(µL) Master Mix(x5.5)(µL) Milli-Q H2O 36.8 185.9 10x Pfu Buffer with MgSO4 5 27.5 dNTPs 2 11 VF2 Primer 2 11 VR Primer 2 11 Template DNA 2 Pfu polymerase 0.2 1.1
Added 45 (µL) MM to each tube.
Analyzed PCR products of BioBrick standard assembly; 3 part (or 3 antibiotic) assembly; and 3 part (3AB) Intermediate/2 part assembly on a 2% TAE agarose gel.
GEL PICTURE!
(In Lab: ADS)
Objective: Produce large quantities of pSB1A3, pSB1C3 and pSB1T3.
Method:
Component 1X(µL) Master Mix(x3.2)(µL) Milli-Q H2O 75 240 10x Pfu Buffer with MgSO4 10 32 dNTPs 5 16 Primer (SB-prep-2Ea) 3.5 11.2 Primer (SB-prep-3P) 3.5 11.2 Template DNA 2 Pfu polymerase 1 3.2
Added 98(µL) to each tube. Used PLASRB PCR Protocol from Aug. 13, 2010.
Aug 16, 2010
(In Lab: KG)
Objective: Confirm overnight ligations done on August 15, 2010.
Method:
Screening via PCR amplification : Thermocycler set to iGEM program 4
Component 1X(µL) Milli-Q H2O 33.8 10x Pfu Buffer with MgSO4 5 dNTPs 2 VF2 Primer 2 VR Primer 2 Template DNA 5 Pfu polymerase 0.2
3AB Master Mix
Component Master Mix(x5.5)(µL) Milli-Q H2O 185.9 10x Pfu Buffer with MgSO4 27.5 dNTPs 11 Prefix Primer 11 Suffix Primer 11 Template DNA 2 Pfu polymerase 1.1
BBS/pSB1C3 Master Mix
Component Master Mix(x11)(µL) Milli-Q H2O 371.8 10x Pfu Buffer with MgSO4 55 dNTPs 22 VF2 Primer 22 VR Primer 22 Template DNA Pfu polymerase 2.2
Analyzed PCR products of overnight BioBrick standard assembly; 3 part (or 3 antibiotic) assembly; and 3 part (3AB) Intermediate/2 part assembly on a 2% TAE agarose gel.
GEL PICTURE!
Aug 16, 2010 Evening
(In Lab: KG, AS)
Objective: Restriction of PCR Products (mms6-dT, lumazine-dT). Restriction is necessary for ligation into plasmid backbone pSB1C3
Method:
Restriction Reactions:
Ingredient 1X(µL) Master Mix(x5.5)(µL) MilliQ H20 Water 7.6 41.8 Orange Buffer (10x) 2 11 pDNA 10 PstI 0.2 1.1 SpeI 0.2 1.1 Incubated at 37oC for 1.5 hours. Heat killed enzymes for 2 minutes at 80oC.
Ligation Reactions:
Ingredient 1X(µL) Master Mix(x5.5)(µL) MilliQ H20 Water 13.8 75.9 T4 Ligase Buffer (10x) 2 11 Plasmid Backbone (pSB1C3) 2 11 pDNA 2 T4 DNA Ligase 0.2 1.1
(In Lab: KG)
Objective: Transformations of insertions of mms6 or lumazine into pET28a.
Method: used Competent Cell Transformation protocol
- changes:
- used 50µL aliquottes of DH5&alpha
- did not pipette up and down once, the cells were just swirled 3 times
- added 400µL SOC media, shoock at 370C for 90 min
- platted 250µL and 150µL
results contents &250µL 150µL + control(pUC19) good good mms6 good good mms6-2 good good mms6 good x Lumazine good x Lumazine good x
Aug 17, 2010
(In Lab: JV)
Objective: Confirm ligations done on August 16, 2010.
Method:
Screening via PCR amplification : Thermocycler set to iGEM program 4
Component 1X(µL) Master Mix(x5.5)(µL) Milli-Q H2O 12.8 70.4 10x Pfu Buffer with MgSO4 2 11 dNTPs 1 5.5 VF2 Primer 1 5.5 VR Primer 1 5.5 Template DNA 2 Pfu polymerase 0.2 1.1
Ran a 2% Agarose gel in 1X TAE buffer for 65 minutes at 100V.
GEL PICTURE!
Aug 17, 2010 Evening
(In Lab: AS)
Objective: Repeat ligation of mms6-dT and lumazine-dT to pSB1C3.
Method: Use already restricted mms6-dT and lumazine-dT. Restrict pSB1C3 PCR product with EcoRI and PstI.
Restriction Reactions:
Ingredient 1X(µL) MilliQ H20 Water 28.6 Orange Buffer (10x) 6 pDNA (pSB1C3) 25 PstI 0.2 EcoRI 0.2 Incubated at 37oC for 1.5 hours. Heat killed enzymes for 20 minutes at 80oC.
Ligation Reactions:
Ingredient 1X(µL) Master Mix(x5.5)(µL) MilliQ H20 Water 13.8 75.9 T4 Ligase Buffer (10x) 2 11 Part 2 (pSB1C3) 2 11 Part 1 (mms6-dT/Lum-dT) 2 T4 DNA Ligase 0.2 1.1 Added 18(µL) MM to each rxn tube. Incubated overnight at room temperature.
Objective: Ligation confirmation by PCR. 2 different PCR reaction conditions were utilized. Believe PstI is not being heat inactivated.
Method:
PCR 1 - Show complete insertion of mms6-dT, lumazine-dT into pSB1C3. Both EcoRI and PstI ligations occurred.
Component 1X(µL) Master Mix(x11)(µL) Milli-Q H2O 33.8 371.8 10x Pfu Buffer with MgSO4 5 55 dNTPs 2 22 Forward VF2 Primer 2 22 Reverse VR Primer 2 22 Template DNA 5 Pfu polymerase 0.2 2.2
Added 45(µL) MM to each rxn tube.
PCR 2 - Show PstI is not heat killed and only EcoRI ligation occurred.
Component 1X(µL) Master Mix(x16)(µL) Milli-Q H2O 33.8 540.8 10x Pfu Buffer with MgSO4 5 80 dNTPs 2 32 Forward VF2 Primer 2 32 Reverse VR Primer 2 32 Template DNA 5 Pfu polymerase 0.2 3.2
Added 45(µL) MM to each rxn tube.
Objective: Analyze tendency of PstI to be heat killed.
Hypothesis: Ligation will be counteracted by PstI digestion even following 20 minute heat kill at 80oC .
Method: Restrict plasmid DNA with PstI (and EcoRI control). Reactions stopped by heat killing. Plasmids re-ligated with T4 DNA Ligase. Ligation confirmed with PCR. If PCR product is present then enzyme heat killed but if there is no PCR Product then enzyme was not heat killed.
Restriction Reactions:
Ingredient 1X(µL) MilliQ H20 Water 12.8 Orange Buffer (10x) 2 pDNA (dT) 5 PstI 0.2 EcoRI (control only) 0.2 Incubated at 37oC for 1.5 hours. Heat killed enzymes for 20 minutes at 80oC.
Ligation Reactions:
Ingredient 1X(µL) MilliQ H20 Water 15.8 T4 Ligase Buffer (10x) 2 Restriction Mix 2 T4 DNA Ligase 0.2 Incubated at room temperature overnight.
PCR to confirm Ligation
Component 1X(µL) Master Mix(x2.2)(µL) Milli-Q H2O 33.8 74.36 10x Pfu Buffer with MgSO4 5 11 dNTPs 2 4.4 Forward VF2 Primer 2 4.4 Reverse VR Primer 2 4.4 Template DNA 5 Pfu polymerase 0.2 0.44
Added 45(µL) to each reaction tube.
Aug 18, 2010
(In Lab: JV)
Objective: Confirmation of ADS' analysis of PstI's tendency to be heat killed.
Method: Will PCR ligation reactions from different assembly methods and EcoRI and PstI controls.
Component 1X(µL) Milli-Q H2O 33.8 10x Pfu Buffer with MgSO4 5 dNTPs 2 Forward VF2 Primer 2 Reverse VR Primer 2 Template DNA 5 Pfu polymerase 0.2
Ran on cycle 4 of thermocycler.
Viewed PCRs on 2% TAE agarose gel that ran at 110 V for 30 minutes.
GEL PICTURE!!!
(In Lab: HB)
Objective: Analyze tendency of PstI to be heat killed.
Method: Restrict plasmid DNA with PstI (and EcoRI control). Reactions stopped by heat killing. Plasmids re-ligated with T4 DNA Ligase. Ligation confirmed with PCR. If PCR product is present then enzyme heat killed but if there is no PCR Product then enzyme was not heat killed.
Restriction Reactions:
Ingredient 1X(µL) MilliQ H20 Water 12.8 Orange Buffer (10x) 2 pDNA (dT) 5 PstI 0.2 EcoRI (control only) 0.2 Incubated at 37oC for 1.5 hours. Heat killed enzymes for 20 minutes at 80oC.
Ligation Reactions:
Ingredient 1X(µL) MilliQ H20 Water 15.8 T4 Ligase Buffer (10x) 2 Restriction Mix 2 T4 DNA Ligase 0.2 Incubated at room temperature overnight.
PCR to Confirm Ligation
Component 1X(µL) Master Mix(x6.5)(µL) Milli-Q H2O 33.8 219.7 10x Pfu Buffer with MgSO4 5 32.5 dNTPs 2 13 Forward VF2 Primer 2 13 Reverse VR Primer 2 13 Template DNA 5 Pfu polymerase 0.2 1.3
Added 45(µL) to each reaction tube. Used thermocycler iGEM Program 4.
3 different treatments: 1. Restricted, heat killed and ligated. 2. Restricted and heat killed. 3. Restricted and not heat killed.
Aug 19, 2010
(In Lab: JV)
Objective: Determine if any transformations from Aug 16, 2010 have the correct insert.
Method: Pick colonies and incubate at 37oC in LB Media with Kan overnight. Use QIAGEN method to extract plasmid DNA. Restrict plasmid DNA to determine if mms6 or lumazine has correctly ligated into pET-28(A).
Restriction Reactions:
mms6 RESTRICTED -
Ingredient 1X(µL) Master Mix(x31)(µL) MilliQ H20 Water 15.75 488.25 Red Buffer (10x) 2 62 Template DNA Enzyme (EcoRV) 0.25 7.75 mms6 UNRESTRICTED -
Ingredient 1X(µL) Master Mix(x31)(µL) MilliQ H20 Water 16 496 Red Buffer (10x) 2 62 Template DNA lumazine RESTRICTED -
Ingredient 1X(µL) Master Mix(x31)(µL) MilliQ H20 Water 15.75 488.25 Tango Buffer (10x) 2 62 Template DNA Enzyme (EcoRV) .25 7.75 lumazine UNRESTRICTED -
Ingredient 1X(µL) Master Mix(x31)(µL) MilliQ H20 Water 16 496 Tango Buffer (10x) 2 62 Template DNA Added 18(µL) to each restriction digest reaction. Incubated at 37oC for 1.5 hours. Heat killed enzymes for 20 minutes at 80oC.
Samples were run on a 2% agarose gel in 1X TAE Buffer.
GEL PICTURE!
(In Lab: HB)
Objective: Run a PCR testing VF2/Suffix and Prefix/VR. A colony PCR of lumazine and mms6 in pET28a. A PCR of 15 ligation tests.
(In Lab: JV)
Objective: Screen for colonies with the correct insert from Aug. 4, 2010 transformations.
Method: Colony PCR. Changes - Used pipette tip instead of toothpick. Put colony in 20(µL) autoclaved Milli-Q water.
PCR: Thermocycler set to iGEM Program 4
Component 1X(µL) Milli-Q H2O 9.8 10x Pfu Buffer with MgSO4 2 dNTPs 2 Forward Primer (VF2) 2 Reverse Primer (VR) 2 Template DNA (Cell Lysate) 2 Pfu polymerase 0.2
Added 18µL Master Mix to each reaction tube.
Method: Control test of primer combinations.
Combination 1 - VF2/Suffix Combination 2 - Prefix/VR Combination 3 - Prefix/Suffix dT maxiprep used as known template DNA source.
PCR Combination 1:
Component 1X(µL) Milli-Q H2O 9.8 10x Pfu Buffer with MgSO4 2 dNTPs 2 Forward Primer (Prefix) 2 Reverse Primer (Suffix) 2 Template DNA (dT) 2 Pfu polymerase 0.2
PCR Combination 2:
Component 1X(µL) Milli-Q H2O 9.8 10x Pfu Buffer with MgSO4 2 dNTPs 2 Forward Primer (Prefix) 2 Reverse Primer (VR) 2 Template DNA (dT) 2 Pfu polymerase 0.2
PCR Combination 3:
Component 1X(µL) Milli-Q H2O 9.8 10x Pfu Buffer with MgSO4 2 dNTPs 2 Forward Primer (VF2) 2 Reverse Primer (suffix) 2 Template DNA (dT) 2 Pfu polymerase 0.2
Method: PCR of 15 ligation tests.
Component 1X(µL) Master Mix(x19.5)(µL) Milli-Q H2O 9.8 191.1 10x Pfu Buffer with MgSO4 2 39 dNTPs 2 39 Forward VF2 Primer 2 39 Reverse VR Primer 2 39 Template DNA 2 Pfu polymerase 0.2 3.9
Added 18(µL) to each tube.
(In Lab: JV)
Analyzed HB's PCR products on 2.% TAE gel which ran for 60 minutes at 100 V. GEL PICTURE!!!Aug 20, 2010
(In Lab: JV)
Objective: Determine if attempts to PCR amplify plasmid backbone were successful.
Method: Ran samples on 1% agarose gel with 1X TAE buffer for 50 minutes at 100 V.
Results: DNA concentration was good, however there was no evidence of an insert into pET-28(A).
GEL PICTURE!!!
Aug 23, 2010
(In Lab: JV)
Objective: Obtained part <partinfo>BBa_K118021</partinfo> and <partinfo>BBa_I716462</partinfo>.
Method: Used competent cell transformation protocol.
(In Lab: HB)
Objective: Restrict 18 maxiprepped parts to quantify DNA.
Method: Restrict all 18 parts and run on a 1% agarose gel with unrestricted parts.
Restriction Reactions:
Restriction Mix -
Ingredient 1X(µL) Master Mix(x19)(µL) MilliQ H20 Water 12.8 243.2 Orange Buffer (10x) 2 38 Template DNA 5 EcoRI 0.2 3.8 Unrestricted Mix -
Ingredient 1X(µL) Master Mix(x19)(µL) MilliQ H20 Water 13 247 Orange Buffer (10x) 2 38 Template DNA 5 15(µL) added to each rxn tube. Incubated at 37oC for 1.5 hours.
GEL PICTURE!
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