Team:Lethbridge/Notebook/Lab Work/August

From 2010.igem.org

(Difference between revisions)
(→Aug 16, 2010 Evening)
(→Aug 16, 2010 Evening)
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<b>Objective:</b> Transformations of insertions of mms6 or lumazine into pET28a. <br>
<b>Objective:</b> Transformations of insertions of mms6 or lumazine into pET28a. <br>
-
<b>Method:</b> Transformation of competent cells protocol.<br>
+
<b>Method:</b> used [[Team:Lethbridge/Notebook/Protocols|Competent Cell Transformation]] protocol
 +
* changes:
 +
**used 50&micro;L aliquottes of DH5&alpha
 +
**did not pipette up and down once, the cells were just swirled 3 times
 +
**added 400&micro;L SOC media, shoock at 37<sup>0</sup>C for 90 min
 +
**platted 250&micro;L and 150&micro;L<br>
-
With the following changes:
+
<table border ="3">
-
1.  50(&micro;L) aliquots of DH5alpha.
+
<td><b>results</b>
-
2.  Added 400(&micro;L) SOC media and put in shaker for 90 minutes at 37<sup>o</sup>C. 
+
<tr><td>contents<td><b>&250&micro;L</b><td><b>150&micro;L</b>
-
3.  Plated 150(&micro;L) and 250(&micro;L).
+
<tr><td>+ control(pUC19)<td>good<td>good
-
 
+
<tr><td>mms6<td>good<td>good
-
<b>Results:</b> .<br>
+
<tr><td>mms6-2<td>good<td>good
 +
<tr><td>mms6<td>good<td>x
 +
<tr><td>Lumazine<td>good<td>x
 +
<tr><td>Lumazine<td>good<td>x
 +
</table><br>
==<font color="white">Aug 17, 2010 ==
==<font color="white">Aug 17, 2010 ==

Revision as of 05:09, 22 October 2010



Feel free to look around our notebook!

Here you can check out the work we have done in the lab, click on a month to take a look!

Contents

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
ConstructpDNAbufferEnzymes
pBAD-sRBS/mRBSpBADRedEcoRI and SpeI
pBAD-sRBS/mRBSsRBSOrangeXbaI and EcoRI
pBAD-sRBS/mRBSmRBSOrangeXbaI and EcoRII
sRBS/mRBS-TetRsRBSRedPstI and SpeI
sRBS/mRBS-TetRmRBSRedPstI and SpeI
sRBS/mRBS-TetRTetRTangoXbaI and PstI
TetR-dTTetRRedEcoRI and SpeI
TetR-dTdTOrangeXbaI and EcoRI
dT-pTetdTRedEcoRI and SpeI
dT-pTetpTetOrangeXbaI and EcoRI
pLAcI-sRBS/mRBSpLacIRedEcoRI and SpeI
pLAcI-sRBS/mRBSsRBSOrangeXbaI and EcoRI
pLAcI-sRBS/mRBSmRBSOrangeXbaI and EcoRI
Mms6-PET28(a)PET28(a)OrangeNotI
  • 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

ComponentVolume per tube (µL)Master Mix (x6)
MilliQ H2O41.8250.8
10x Pfu Buffer with MgSO4530
dNTPs16
Forward Primer0.53
Reverse Primer0.53
Template DNA1-
Pfu DNA polymerase0.2-
Total50292.8
  • 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:

LaneSampleComponents (µL)
11 kb ladder2 loading dye (5x) + 0.5 ladder + 3.5 MilliQ H2O
2ECFP2 DNA + 2 loading dye (5x) + 2 MilliQ H2O
3EYFP2 DNA + 2 loading dye (5x) + 2 MilliQ H2O
4Lumazine2 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:

RestrictionMilliQ H2O (µL)Buffer Orange (µL)pDNA (µL)Enzyme (µL)
pSB1C379.2510100.25 EcoRI + 0.25 PstI + 0.25 DpnI
pSB1C3 control801010-
dT79.5010100.25 EcoRI + 0.25 PstI
dT control801010-
  • 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.

LaneContentsResult
11kb ladder
2pTet (XbaI, EcoRI)good
3pTet (orange buffer)---
4dT (XbaI, EcoR)no good
5dT (orange buffer)---
6mRBS (SpeI, PstI) good
7mRBS (red buffer)---
8sRBS (SpeI, PstI) good
9sRBS (red buffer)---
10mRBS (XbaI, EcoRI) good
11mRBS (orange buffer)---
12sRBS (XbaI, EcoRl) good
13sRBS (orange buffer)---
14pet28(a) good
15100 bp ladder
16PSB1C3 not good
17PSB1C3 restriction digest---

LaneContentsResult
1TetR (EcorI, SpeI) good
2TetR (red buffer)---
3pLacI (EcoRI, SpeI) good
4pLacI (red buffer)---
5Mms6can not tell
6Mms6 control---
7TetR (Xbal, PstI) good
8TetR (tango buffer)---
9pBAD (EcoRI, SpeI)good
10pBAD (red buffer)---
11dT (EcoRI, SpeI)good
12dT (red buffer)---
13pLacI (2)?
14dT control not good
15dT restriction
16100 bp ladder
17dT PCR product good
18Mms6 PCR product good
19pBAD PCR product good
20pLacI 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

Component1X(µL)Master Mix(x16)(µL)
Milli-Q H2O41.8668.6
10x Pfu Buffer with MgSO4580
dNTPs116
Forward Primer0.58
Reverse Primers0.58
Template DNA116
Pfu polymerase0.23.2

2.5% agarose gel(1x TAE)

lanecontentsSuccessful Ligation ?
150bp ladder---
2dt pSBIC3---
3dt pTetx
4dt control---
5sRBS-TetRx
6mRBS-TetR?
7TetR control---
8pLacI-mRBSx
9pLacI-sRBS?
10pLacI control---
11Mms6 pET-28ano band
12Mms6 control---
13pBad-SRBSx
14pBad-mRBSx
15pBad 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&250µL150µL
dt-pTetgoodx
- controlxx
mms6-pET-28agoodgood
dt-pSBIC3xx
mRBS-TetRgoodgood
pLacI-mRBSgoodgood
SRBS-TetRxx
pBAD-SRBSgoodgood
+ contolgoodgood

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:

RestrictionMilliQ H2O (µL)Buffer Orange (µL)pDNA (µL)Enzyme (µL)
mms6799.51001001 NotI

PCR:

Component1X(µL)
Milli-Q H2O41.85
10x Pfu Buffer with MgSO45
dNTPs1
Forward Primer (VF2)0.5
Reverse Primers (VR)0.5
Template DNA (Lumazine Synthase)1
Pfu polymerase0.2

2% Agarose Gel for Gel Extraction of mms6:

lanesampleloaded
1mms6 restricted with NotI1 mL sample
2mms6 unrestricted control10(µL) sample, 2(µL)dye
350bp ladder2(µL) ladder, 2(µL) dye, 8(µL) H20
31 kb ladder2(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'dEndy cont'd
setup 20(µL) reactionsetup 20(µL) reaction
1(µL) colony suspension2(µL) colony suspension
2(µL) 10x p.fu (+Mg SO42(µL) 10x p.fu (+Mg SO4
2(µL) dNTP2(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 polymerase0.2(µL) Pfu polymerase
11.8 Milli-Q H2O12.6 Milli-Q H2O

  • as control for each rxn used equal volume of mRBS maxiprep
Knight cont'dEndy cont'd
cycling conditionscycling conditions
950C for 15 minutes950C 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 minutes700C 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.

lanesampleloaded
150bp ladder1(µL) ladder, 1(µL) dye, 4(µL) H20
2Knight control5(µL) sample, 1(µL)dye
3Knight colony5(µL) sample, 1(µL)dye
4Endy control5(µL) sample, 1(µL)dye
5Endy colony5(µL) sample, 1(µL)dye
650bp ladder1(µL) ladder, 1(µL) dye, 4(µL) H20
7lumazine(justin's)5(µL) sample, 1(µL)dye

Repeat gel with template controls

lanesampleloaded
150bp ladder0.5(µL) ladder, 2(µL) dye, 9.5(µL) H20
2Endy Template5(µL) colony suspension, 1(µL)dye, 4(µL) H20
3Endy mRBS Control (PLR)5(µL) sample, 1(µL)dye
4Endy mRBS-TetR colony(PCR)5(µL) sample, 1(µL)dye
4mRBS template0.5(µL) sample, 1(µL)dye, 4(µL) H20
5Knight Template0.25(µL) colony suspension, 1(µL)dye, 4(µL) H20
6Knight mRBS control5(µL) ladder, 1(µL) dye
7Knight-mRBS-TetR colony5(µL) sample, 1(µL)dye
11Kb ladder0.5(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

Component1X(µL)Master Mix(x4)(µL)
Milli-Q H2O41.8167.2
10x Pfu Buffer with MgSO4520
dNTPs14
Forward Primer (VF2)0.52
Reverse Primers (VR)0.52
Template DNA1
Pfu polymerase0.2

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.

lanesampleloaded
150bp ladder0.5(µL) ladder, 1(µL) dye, 6.5(µL) H20
2mRBS-xylE I15(µL) sample, 2(µL)dye
3mRBS-xylE I25(µL) sample, 2(µL)dye
4Lumazine5(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)

IngredientVolume(µL)
MilliQ H20 Water15.75
Orange Buffer (10x)2
pDNA2
EcoRI0.25


Restriction Reaction (DOUBLE)

IngredientVolume(µL)
MilliQ H20 Water15.50
Orange Buffer (10x)2
pDNA2
EcoRI0.25
PstI0.25


Unrestricted Control

IngredientVolume(µL)
MilliQ H20 Water16
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:

LaneContents
11kb ladder
250 bp ladder
3dT-pTet 1 DRD
4dT-pTet 1 SRD
5dT-pTet 1 URD
6dT-pTet 3 DRD
7dT-pTet 3 SRD
8dT-pTet 3 URD
9pLacI-mRBS 1 DRD
10pLacI-mRBS 1 SRD
11pLacI-mRBS 1 URD
12pLacI-mRBS 2 DRD
13pLacI-mRBS 2 SRD
14pLacI-mRBS 2 URD
15pLacI-sRBS 1 DRD
16pLacI-sRBS 1 SRD
17pLacI-sRBS 1 URD
18pLacI-sRBS 3 DRD
19pLacI-sRBS 3 SRD
20pLacI-sRBS 3 URD

LaneContents
11 kb ladder
250 bp ladder
3pBAD-mRBS 1 DRD
4pBAD-mRBS 1 SRD
5pBAD-mRBS 1 URD
6pBAD-mRBS 2 DRD
7pBAD-mRBS 2 SRD
8pBAD-mRBS 2 URD
9pBAD-sRBS 1 DRD
10pBAD-sRBS 1 SRD
11pBAD-sRBS 1 URD
12pBAD-sRBS 2DRD
13pBAD-sRBS 2 SRD
14pBAD-sRBS 2 URD
15mRBS-TetR 1 DRD
16mRBS-TetR 1 SRD
17mRBS-TetR 1 URD
18mRBS-TetR 3 DRD
19mRBS-TetR 3 SRD
20mRBS-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:
    ComponentVolume (µL)
    MilliQ H2O15.6 or 15.8
    Buffer2
    pDNA2
    Enzyme0.20 + 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:

    LaneContents
    150 bp ladder
    2pBAD-mRBS 2 URD
    3pBAD-mRBS 2 SRD
    4pBAD-mRBS 2 DRD
    5pLacI-sRBS 3 URD
    6pLacI-sRBS 3 SRD
    7pLacI-sRBS 3 DRD
    8pLacI-mRBS 2 URD
    9pLacI-mRBS 2 SRD
    10pLacI-mRBS 1 DRD
    11dT-pTet 3 URD
    12dT-pTet 3 SRD
    13dT-pTet 3 DRD
    14pBAD-mRBS 1 URD
    15pBAD-mRBS 1 SRD
    16pBAD-mRBS 1 DRD
    17pLacI-sRBS 2 URD
    18pLacI-sRBS 2 SRD
    19pLacI-sRBS 2 DRD
    201 kb ladder

    LaneContents
    150 bp ladder
    2pLacI-mRBS 1 URD
    3pLacI-mRBS 1 SRD
    4pLacI-mRBS 1 DRD
    5dT-pTet 1 URD
    6dT-pTet 1 SRD
    7dT-pTet 1 DRD
    8mRBS-TetR 3 URD
    9mRBS-TetR 3 SRD
    10mRBS-TetR 3 DRD
    11mRBS-TetR 1 URD
    12mRBS-TetR 1 SRD
    13mRBS-TetR 1 DRD
    14pBAD-sRBS 2 URD
    15pBAD-sRBS 2 SRD
    16pBAD-sRBS 2 DRD
    17pBAD-sRBS 1 URD
    18pBAD-sRBS 1 SRD
    19pBAD-sRBS 1 DRD
    201 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

    Component1X(µL)Master Mix(x65)(µL)
    Milli-Q H2O10.9708.5
    5X Phusion HF Buffer4260
    dNTPs165
    Forward Primer (VF2)165
    Reverse Primers (VR)165
    Colony Template2
    Phusion polymerase0.1711.05

    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.

    Component1X(µL)Master Mix(x6.5)(µL)
    Milli-Q H2O8.857.2
    5X Phusion HF Buffer426
    dNTPs213
    Forward Primer 213
    Reverse Primer213
    Template DNA16.5
    Polymerase0.21.3

    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

    Component1X(µL)Master Mix(x7.5)(µL)
    Milli-Q H2O9.873.5
    10x Pfu Buffer with MgSO4215
    dNTPs215
    Forward Primer (VF2)215
    Reverse Primer (VR)215
    Template DNA2
    Pfu polymerase0.21.5

    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

    Component1X(µL)Master Mix(x47)(µL)
    Milli-Q H2O9.8460.6
    10x Pfu Buffer with MgSO4294
    dNTPs294
    Forward Primer (VF2)294
    Reverse Primer (VR)294
    Template DNA (Cell Lysate)294
    Pfu polymerase0.29.4

    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)

    Component1X(µL)Master Mix(x16.5)(µL)
    Milli-Q H2O9.8161.7
    10x Pfu Buffer with MgSO4233
    dNTPs233
    Forward Primer (Prefix)233
    Reverse Primer (Suffix Antisense)233
    Template DNA (Cell Lysate)2
    Pfu polymerase0.23.3

    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.


    Component1X(µL)Master Mix(x12.5)(µL)
    Milli-Q H2O41.85523.1
    10x Pfu Buffer with MgSO4562.5
    dNTPs112.5
    Forward Primer (VF2)0.56.25
    Reverse Primer (VR)0.56.25
    Template DNA1
    Pfu polymerase0.151.888

    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.

    Component1X(µL)Master Mix(x13.5)(µL)
    Milli-Q H2O14.952.15
    10x Pfu Buffer with MgSO427
    dNTPs13.5
    Forward Primer (SB-prep-2)0.72.45
    Reverse Primer (SB-prep-3p)0.72.45
    Template DNA0.5
    Pfu polymerase0.20.7

    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)

    lanecontents
    1pBAD-mRBS 1
    2pBAD-mRBS 2
    3pBAD-sRBS 1
    4pBAD-sRBS 2
    5mRBS-TetR 1
    6mRBS-TetR 3
    750 bp Ladder
    8dT-pTet 1
    9dT-pTet 3
    10pLacI-mRBS 1
    11pLacI-mRBS 2
    12pLacI-sRBS 2
    13pLacI-sRBS 3
    14MT
    15MT
    16MT
    17MT
    18MT
    19MT
    20MT
    21No Lanes
    22No Lanes
    23No Lanes
    24No Lanes
    25No Lanes
    26No Lanes
    27No Lanes
    lanecontents
    1K249001
    2K249004
    3K249005
    4K249006
    5MT
    6K249008
    7K249008 (Qiagen)
    8K249014
    9K249017
    1050 bp Ladder
    111
    122
    133
    144
    155
    16xylE-dT
    17Lumazine-dT
    18pLacI-sRBS
    19MT
    20MT
    21MT
    22MT
    23MT
    24MT
    25MT
    26MT
    27MT

    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 -

    Ingredient1X(µL)Master Mix(x8.5)(µL)
    MilliQ H20 Water7.866.30
    Orange Buffer (10x)217
    pDNA10
    NotI0.21.7

    Added 10 (µL) to each tube.

    For pET28a -

    IngredientReaction Mix(µL)
    MilliQ H20 Water4.8
    Orange Buffer (10x)5
    pDNA40
    NotI0.2

    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

    Component1X(µL)Master Mix(x10.5)(µL)
    Milli-Q H2O10.8113.4
    10x Pfu Buffer with MgSO4221
    dNTPs221
    Forward Primer (Prefix)221
    Reverse Primers (Suffix Antisense)221
    Template DNA1
    Pfu polymerase0.22.1

    Added 19 (µL) to each tube.

    Restriction Reactions:

    For lumazine and mms6 -

    Ingredient1X(µL)Master Mix(x5.5)(µL)
    MilliQ H20 Water11.663.8
    Red Buffer (10x)211
    pDNA6
    EcoRI0.21.1
    SpeI0.21.1

    Added 14 (µL) to each tube.

    For dT -

    IngredientReaction Mix(µL)
    MilliQ H20 Water58
    Tango Buffer (10x)10
    pDNA30
    XbaI1
    PstI1

    For psB1C3 -

    IngredientReaction Mix(µL)
    MilliQ H20 Water70
    Orange Buffer (10x)10
    pDNA30
    EcoRI1
    PstI1
    DpnI1

    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 -

    Ingredient1X(µL)Master Mix(x5.5)(µL)
    MilliQ H20 Water15.886.9
    Tango Buffer (10x)211
    pDNA2
    SpeI0.21.1

    Added 18 (µL) to each reaction.

    For dT, CUT with XbaI-

    IngredientReaction Mix(µL)
    MilliQ H20 Water79
    Tango Buffer (10x)10
    pDNA10
    XbaI1

    Incubated at 37oC for 1.5 hours. Heat killed enzymes for 20 minutes at 80oC.

    Ligation Reactions:

    3 Part: Lumazine/mms6 + dT + psB1C3

    Ingredient1X(µL)Master Mix(x5.5)(µL)
    MilliQ H20 Water11.064.9
    T4 Ligase Buffer (10x)211
    Plasmid (psB1C3)211
    Part 1 (Lumazine/mms6)2
    Part 2 (dT)211
    T4 DNA Ligase0.21.1

    2 Part: Lumazine/mms6 + dT

    Ingredient1X(µL)Master Mix(x5.5)(µL)
    MilliQ H20 Water13.875.9
    T4 Ligase Buffer (10x)211
    Part 1 (Lumazine/mms6)2
    Part 2 (dT)211
    T4 DNA Ligase0.21.1

    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

    Component1X(µL)Master Mix(x5.5)(µL)
    Milli-Q H2O33.8185.9
    10x Pfu Buffer with MgSO4527.5
    dNTPs211
    VF2 Primer211
    VR Primer211
    Template DNA5
    Pfu polymerase0.21.1

    Added 45 (µL) MM to each tube.

    2 Part: Lum/mms6 + dT

    Component1X(µL)Master Mix(x5.5)(µL)
    Milli-Q H2O6.837.4
    10x Pfu Buffer with MgSO4211
    dNTPs211
    Prefix Primer211
    Suffix Antisense Primer211
    Template DNA5
    Pfu polymerase0.21.1

    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 -

    Ingredient1X(µL)Master Mix(x10.5)(µL)
    MilliQ H20 Water11.8123.9
    Orange Buffer (10x)221
    pDNA6
    NotI0.22.1

    Added 14 (µL) to each tube.

    For pET28a -

    IngredientReaction Mix(µL)
    MilliQ H20 Water4.8
    Orange Buffer (10x)5
    pDNA40
    NotI0.2

    Incubated at 37oC for 1.5 hours. Heat killed enzymes at 80oC for 20 minutes.

    Ligation Reactions:

    Ingredient1X(µL)Master Mix(x5.5)(µL)
    MilliQ H20 Water13.975.9
    T4 Ligase Buffer (10x)211
    Plasmid (pET28a)211
    Cut out part (Lumazine/mms6)2
    T4 DNA Ligase0.21.1

    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 -

    Ingredient1X(µL)Master Mix(x5.5)(µL)
    MilliQ H20 Water7.641.8
    Red Buffer (10x)211
    pDNA10
    EcoRI0.21.1
    SpeI0.21.1

    For dT -

    IngredientReaction Mix(µL)
    MilliQ H20 Water38
    Orange Buffer (10x)10
    pDNA50
    XbaI1
    EcoRI1

    Incubated at 37oC for 1.5 hours. Heat killed enzymes for 20 minutes at 80oC.

    Ligation Reactions:

    Ingredient1X(µL)Master Mix(x5.5)(µL)
    MilliQ H20 Water13.875.9
    T4 Ligase Buffer (10x)211
    Plasmid (dT)211
    Cut out part (Lumazine/mms6)2
    T4 DNA Ligase0.21.1

    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

    Component1X(µL)Master Mix(x5.5)(µL)
    Milli-Q H2O36.8185.9
    10x Pfu Buffer with MgSO4527.5
    dNTPs211
    VF2 Primer211
    VR Primer211
    Template DNA2
    Pfu polymerase0.21.1

    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

    Component1X(µL)
    Milli-Q H2O33.8
    10x Pfu Buffer with MgSO45
    dNTPs2
    VF2 Primer2
    VR Primer2
    Template DNA5
    Pfu polymerase0.2

    3AB Master Mix

    ComponentMaster Mix(x5.5)(µL)
    Milli-Q H2O185.9
    10x Pfu Buffer with MgSO427.5
    dNTPs11
    Prefix Primer11
    Suffix Primer11
    Template DNA2
    Pfu polymerase1.1

    BBS/pSB1C3 Master Mix

    ComponentMaster Mix(x11)(µL)
    Milli-Q H2O371.8
    10x Pfu Buffer with MgSO455
    dNTPs22
    VF2 Primer22
    VR Primer22
    Template DNA
    Pfu polymerase2.2

    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:

    Ingredient1X(µL)Master Mix(x5.5)(µL)
    MilliQ H20 Water7.641.8
    Orange Buffer (10x)211
    pDNA10
    PstI0.21.1
    SpeI0.21.1

    Incubated at 37oC for 1.5 hours. Heat killed enzymes for 2 minutes at 80oC.

    Ligation Reactions:

    Ingredient1X(µL)Master Mix(x5.5)(µL)
    MilliQ H20 Water13.875.9
    T4 Ligase Buffer (10x)211
    Plasmid Backbone (pSB1C3)211
    pDNA2
    T4 DNA Ligase0.21.1

    (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&250µL150µL
    + control(pUC19)goodgood
    mms6goodgood
    mms6-2goodgood
    mms6goodx
    Lumazinegoodx
    Lumazinegoodx

    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

    Component1X(µL)Master Mix(x5.5)(µL)
    Milli-Q H2O12.870.4
    10x Pfu Buffer with MgSO4211
    dNTPs15.5
    VF2 Primer15.5
    VR Primer15.5
    Template DNA2
    Pfu polymerase0.21.1

    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:

    Ingredient1X(µL)
    MilliQ H20 Water28.6
    Orange Buffer (10x)6
    pDNA (pSB1C3)25
    PstI0.2
    EcoRI0.2

    Incubated at 37oC for 1.5 hours. Heat killed enzymes for 20 minutes at 80oC.

    Ligation Reactions:

    Ingredient1X(µL)Master Mix(x5.5)(µL)
    MilliQ H20 Water13.875.9
    T4 Ligase Buffer (10x)211
    Part 2 (pSB1C3)211
    Part 1 (mms6-dT/Lum-dT)2
    T4 DNA Ligase0.21.1

    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.

    Component1X(µL)Master Mix(x11)(µL)
    Milli-Q H2O33.8371.8
    10x Pfu Buffer with MgSO4555
    dNTPs222
    Forward VF2 Primer222
    Reverse VR Primer222
    Template DNA5
    Pfu polymerase0.22.2

    Added 45(µL) MM to each rxn tube.

    PCR 2 - Show PstI is not heat killed and only EcoRI ligation occurred.

    Component1X(µL)Master Mix(x16)(µL)
    Milli-Q H2O33.8540.8
    10x Pfu Buffer with MgSO4580
    dNTPs232
    Forward VF2 Primer232
    Reverse VR Primer232
    Template DNA5
    Pfu polymerase0.23.2

    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 -

    Ingredient1X(µL)Master Mix(x31)(µL)
    MilliQ H20 Water15.75488.25
    Red Buffer (10x)262
    Template DNA
    Enzyme (EcoRV)0.257.75

    mms6 UNRESTRICTED -

    Ingredient1X(µL)Master Mix(x31)(µL)
    MilliQ H20 Water16496
    Red Buffer (10x)262
    Template DNA

    lumazine RESTRICTED -

    Ingredient1X(µL)Master Mix(x31)(µL)
    MilliQ H20 Water15.75488.25
    Tango Buffer (10x)262
    Template DNA
    Enzyme (EcoRV).257.75

    lumazine UNRESTRICTED -

    Ingredient1X(µL)Master Mix(x31)(µL)
    MilliQ H20 Water16496
    Tango Buffer (10x)262
    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 -

    Ingredient1X(µL)Master Mix(x19)(µL)
    MilliQ H20 Water12.8243.2
    Orange Buffer (10x)238
    Template DNA5
    EcoRI0.23.8

    Unrestricted Mix -

    Ingredient1X(µL)Master Mix(x19)(µL)
    MilliQ H20 Water13247
    Orange Buffer (10x)238
    Template DNA5

    15(µL) added to each rxn tube. Incubated at 37oC for 1.5 hours.

    GEL PICTURE!

    Retrieved from "http://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work/August"