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!
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 | 22
|
VR | 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