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
- Restrict Lumazine wit EcoRI and SpeI (Red Buffer)
- Restrict the dT with XbaI and EcoRI (Orange Buffer)
- Restrict Lumazine Synthase with NotI (Red Buffer)
Set up reactions as follows:
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:
- T4 DNA ligase - 0.25µL
- DNA 1 - 8µL
- DNA 2 - 8µL
- 10x Ligation Buffer - 2µL
- MilliQ H2O - 1.75µL
Incubated reactions overnight at room temperature.
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. 95 for 15 min
2. 98 for 20 sec
3. 55 for 40 sec
4. 72 for 2 min
5. 72 for 20 min
6. 4 infinitely
Aug 13, 2010
(In Lab: AS)
Objective: PCR amplify minipreps prepared on Aug 9/2010 to screen for properly assembled BioBricks.
Method:
PCR: Thermocycler set to iGEM program 7
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 Primers (VR) | 0.5 | 6.25
|
Template DNA | 1 |
|
Pfu polymerase | 0.15 | 1.888
|
Added 49µL Master Mix to each reaction tube.
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!
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 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!
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 pSB1C#
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
|
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.
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!
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!