Team:Lethbridge/Notebook/Lab Work/May

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*Positive control (pUC19) - 9 colonies <br>
*Positive control (pUC19) - 9 colonies <br>
*mms6 (B6) + dT - 1 colonies <br><br>
*mms6 (B6) + dT - 1 colonies <br><br>
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Latest revision as of 01:22, 24 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

May

May 5/2010

(in the lab: JV)
Objective: Test Restriction Endonucleases for activity (take 2)
Relevant Information:
Plasmid DNA used here will be "ES-pSB-CEYFP" from last year's plasmid stocks
Prefix Enzymes are: EcoRI and XbaI
Suffix Enyzmes are: SpeI and PstI
(JV worked out in lab notebook which buffers would be best for each prefix/suffix enzyme combination)
Reactions will be assembled as follows:

EnzymeBufferVolume MM(µL)Volume Enzyme(µL)
PstIRed19.750.25
XbaITango19.750.25
SpeITango19.750.25
EcoRIRed19.750.25
EcoRI/SpeIRed19.50.25 + 0.25
XbaI/SpeITango19.50.25 + 0.25
EcoRI/PstIRed19.50.25 + 0.25
XbaI/PstITango19.50.25 + 0.25

Make up Master Mixes as follows:

Red MMper tube(µL)Total*(µL)
MilliQ H2015.7586.675
Red Buffer (10x)211
pDNA**211

Tango MMper tube(µL)Total*(µL)
MilliQ H2015.7586.675
Tango Buffer (10x)211
pDNA**211
  • Volume per reaction multiplied by 5.5
    • Unknown concentration of pDNA

Incubated for 70min at 37oC (Start-1:05pm; End-2:15pm)
Added 3.3µL of 6x loading dye to each reaction mixture and loaded 10µL onto a 1% agarose gel (in TAE)
Added 1µL of 6x loading dye to 2µL of gene ruler 1kb ladder
Load order as follows:

LaneSampleVolume Loaded (µL)
1pSB-CEYFP/PstI10
2pSB-CEYFP/EcoRI10
3pSB-CEYFP/EcoRI/PstI10
4pSB-CEYFP/EcoRI/SpeI10
5pSB-CEYFP/XbaI/PstI10
6pSB-CEYFP/XbaI10
7pSB-CEYFP/SpeI10
8pSB-CEYFP/XbaI/SpeI10
9pSB-CEYFP/Red Master Mix Control10
10pSB-CEYFP/Tango Master Mix Control10
11pSB-CEYFP/MilliQ H20 Control10
12Ladder4

Ran gel at 100V for 1 hour

Results:

100505JV-EnzymeTest1Cropped.jpg

This gel shows that SpeI does not cut on its own, and does not cut when combined with other enzymes
Conclusion: Test other source of SpeI to see if it has any activity.

May 6/2010

(in the lab: KG, AS)
Objective: To check if the old SpeI enzyme (exp date: March 2011) will cleave plasmid DNA, since we believe the newer SpeI enzyme (exp date: 2012) does not.
Method:

Red Master Mixper tube (µL)Total Volume*
MilliQ H20 Water15.7563
Red Buffer (10x)28
pDNA**28
  • Volume per tube multiplied by 4
    • Used pSB NEYFP pDNA from cell E5 in plasmid box

Enzymes that will use Red Master Mix are: EcoRI+SpeI (old), EcoRI+SpeI (new)
Add 0.25µL of each enzyme to 19.5µL of master mix

Tango Master Mixper tube (µL)Total Volume*
MilliQ H20 Water15.7594.5
Tango Buffer (10x)212
pDNA**212
  • Volume per tube multiplied by 6
    • Used pSB NEYFP pDNA from cell E5 in plasmid box

Enzymes that will use Tango Master Mix are: SpeI (old), SpeI (new), XbaI+SpeI (old), XbaI+SpeI (new)
Add 0.25µL of each enzyme to 19.5µL of master mix

Incubated all reactions at 37oC for 1h (Start-8:30pm; End-9:30pm)
Will not be able to run on agarose gel tonight, will label them so JV can run them in the morning
Tube Names:
Master Mix 1 Control (Red Buffer)
Master Mix 2 Control (Tango Buffer)
E+S(N); EcoRI + SpeI(N)
E+S(O); EcoRI + SpeI(O)
X+S(N); XbaI + SpeI(N)
X+S(O); XbaI + SpeI(O)
S(N); SpeI(N)
S(O); SpeI(O)

Placed in -20oC freezer of later analysis by agarose electrophoresis

May 10/2010

(in the lab:JV)
Objective: To analyze the restriction test done by KG and AS on May 6/2010 by agarose electrophoresis

Method:

LaneSampleQuantity Loaded (µL)
1MM1 Control10
2MM2 Control10
3EcoRI+SpeI(N)10
4EcoRI+SpeI(O)10
5SpeI(N)10
6SpeI(O)10
7XbaI+SpeI(N)10
8XbaI+SpeI(O)10
91kb Ladder5

Run gel for 60min at 100V

Results:

100510JRV-EnzymeTest1Cropped.jpg

It appears as though both SpeI enzymes are working properly here. We will utilize the newer batch of SpeI (expires 2012) from this point forward.

Objective:Make 24 LB agar plates with 100µg/mL ampicillin antibiotic.(JV,KG,AV)
Method:Make 2L of LB media with agar
2x10g Tryptone
2X2.5g Yeast Extract
2x5g NaCl
2x10g Agar

Continued May 11/2010
 (Stock Ampicillin solution is 100mg/mL)
Have 4x500mL of LB with Agar
Add 500µL of stock ampicillin to 500mL of media

May 11/2010 Evening

(in the lab: KG, AV, MC, TF, JV, JS)
Objective: To transform the following plasmids into DH5α E.coli cells.

Construct Name (2009)</td>Construct Location (2009)
LumazineJ4
Lumazine-dTJ5,J6
sRBS-Lumazine-dTJ7,J8
pBAD-TetRI4
pBADA5,F10
sRBSD5,E10
pSB-CEYFPE5,D6
pSB-NEYFPF5,C6
C-term TagC10
N-term TagD9,D10
pTetE4
EYFPA4
CFP CompleteD4

Method: Followed Competent Cell Transformation protocol in Common Protocols section and plated on LB agar supplemented with ampicillin.
Results: The following plasmids were successfully transformed and formed colonies:

  • Lumazine (J4)
  • sRBS-Lumazine-dT (J7)
  • sRBS-Lumazine-dT (J8)
  • pBAD (A5)
  • pBAD (F10)
  • pSB-CEYFP
  • pSB-NEYFP
  • N-term tag
  • EYFP (A4)
  • CFP Complete (D4)

Conclusion: Need another attempt to transform the following plasmids:

  • Lumazine-dT (J5,J6)
  • pBAD-TetR
  • sRBS (D5,E10)
  • C-Term tag
  • pTet

May 12/2010

(in the lab: JV)
Objective: Miniprep of plasmid DNA from transformed cells(JV, AV, HB)
Method:

  • Inoculate 5mL of LB liquid media (with 100µL/mL Ampicillin) with cells from competent cells plates (picked with sterile toothpick).
  • Allow cells in liquid culture to grow overnight in 37oC shaking incubator (300RPM) Purify plasmid DNA from cells by using "Boiling Lysis Plasmid Preparation" protocol in Common Protocols Section.
  • CHANGE: Step 14, used MilliQ H2O (with 20ng/µL RNase A) instead of TE buffer.

Plasmids were transferred to the "iGEM 2010 - Working Plasmid DNA" box in the -20oC freezer in the iGEM lab. Plasmids were placed in the following cells:

ConstructCell in Working Plasmid Box (2010)Original Cell in Old Box
sRBS-Lumazine-dTA1J7
sRNS-Lumazine-dTA2J8
CFP CompleteB6D4
LumazineA3J4
pBADA4A5
pBADA5F10
pSB-CEYFPB5
pSB-NEYFPB4
EYFPB1A4
N-term tagB2

Also generated sterile glycerol stocks and placed in -80oC freezer in the 2010 iGEM box as follows:

ConstructCell Working Glycerol Stock Box (2010)
sRBS-Lumazine-dT (J7)B2,C4,D2
sRNS-Lumazine-dT (J8)C6
CFP CompleteA10, C8
LumazineA8,B10
pBAD (from A5)B5,B9
pBAD (from F10)B3,B7
pSB-CEYFPC3,B5
pSB-NEYFPB6,C1
EYFPC7,B8
N-term tagC2,D4

Objective: Restrict plasmid DNA with restriction endonucleases (JV)
Method:
Have: 10 lanes of restricted plasmid DNA
10 lanes of unrestricted plasmid DNA
1 lane of buffer control
Use EcoRI (prefix cutter) and PstI (suffix cutter)

Pipetting Scheme for Restriction Tubes:

IngredientVolume/tube (µL)Total Volume*
MilliQ H2O15.5155
Red Buffer (10X)220
EcoRI0.252.5
PstI0.252.5
  • Amount per tube multiplied by 10

Pipetting Scheme for Unrestricted reactions:

IngredientVolume/tube (µL)Total Volume*
MilliQ H2O16160
Red Buffer (10X)220
  • Amount per tube multiplied by 10

Buffer Control will be 18µL MilliQ H2O + 2µL 10x Red Buffer.
Place in 37oC water bath at 2:55pm and removed at 4:57pm for a 2 hour incubation.
Analyzed restriction digests on a 1% agarose gel (large gel apparatus ~70mL)
Added 1µL of 6x DNA loading dye to 5µL of sample
Added 2µL of 6x DNA loading dye to 6µL of TAE buffer and 2µL of 1kb DNA mass ladder.
Loaded samples as follows:

Lane</td>Sample</td>Volume Loaded (µL)
11 kb Ladder5
2Buffer Control5
3pSB-NEYFP5
4Restricted Lumazine5
5Lumazine5
6Restricted pSB-NEYFP5
7pSB-CEYFP5
8Restricted pSB-CEYFP5
9pBAD5
10Restricted pBAD5
11EYFP5
12Restricted EYFP5
13CFP Complete5
14Restricted CFP Complete5
15sRBS-Lumazine-dT (J7)5
16Restricted sRBS-Lumazine-dT (J7)5
17N-term Tag5
18Restricted N-term Tag5
19sRBS-Lumazine-dT (J8)5
20Restricted sRBS-Lumazine-dT (J8)5

Ran gel at 100V for 90 minutes (Start-9:50pm; End-11:20pm)
Stained with ethidium bromide for 20 minutes.

Results:

100513TF.KG.JS-Plasmids for Seq.Cropped.jpg

May 13/2010 Evening(in lab: AS,TF,KG,JS,MC)
Objective: To make a second attempt at transforming plasmids that didn't transform the first time. These plasmids are:

  • Lumazine-dT (J5,J6)
  • pBad-TetR
  • sRBS (D5,E10)
  • C-term tag
  • pTet

All DH5α cells were used up in the last transformation, had to aliquot an additional 50x 20µL aliquots (MC,TF)
Transform plasmid DNA (Using "Competent Cell Transformation" Protocol) into newly aliquotted DH5α cells. (KG,JS)
NOTES:
AS concerned that there is something not quite right with LB liquid media added to transformed cells, but continued anyways (JV informed AS the next day that the LB liquid media had not been sterilized).
Plated all 250µL of culture.
Results:

Construct</td>Result
Lumazine-dT(1)Growth present
sRBS-Lumazine-dTGrowth present
sRBS (D5)Growth present
sRBS (E10)Growth present
C-term tagNo growth present
pTetNo growth present

Next Steps:
Make another attempt to transform the C-term tag and pTet constructs.
Start overnight cultures of cells that grew for plasmid prep and sequencing.

May 14/2010

(in the lab: JV)
Objective: Quantify pDNA concentration in order to ensure sufficient material for sequence analysis.
Method: Measure absorbance of samples at 260nm.
Results:

Sample</td>Absorbance at 260nm
sRBS-Lumazine-dT (J7)0.311
sRBS-Lumazine-dT (J8)0.309
CFP complete0.316
N-term tag0.290
pSB-CEYFP0.338
pSB-NEYFP0.403
pBAD (A5)0.282
pBAD (F10)0.562
EYFP0.389
Lumazine0.221

Conclusion: All plasmids present in sufficient concentrations for sequence analysis.

Objective: Purify plasmid DNA from cells recently transformed.
Method:

  • Inoculate 5mL of sterile LB liquid media (with 100µg/mL ampicillin) with cells picked from colonies of transformation plates, including the following:
    Lumazine-dT (J5)
    pBad-TetR
    sRBS (D5,E10)
  • NOTE: Lumazine-dT did NOT grow overnight
  • Followed "Boiling Lysis Plasmid Preparation (Miniprep)" protocol. (May 15/2010; JV,TF)
    NOTE: Added 50µL of MilliQ H2O (with RNase A at a concentration of 20ng/µL) to dissolve pDNA instead of TE buffer.

Objective: Perform restriction digest on the above prepared plasmid DNA.
Method:
Used EcoRI as prefix cutter and PstI as suffix cutter.
Pipetting Scheme for Restriction Tubes:

IngredientVolume/tube (µL)Total Volume*
MilliQ H2O1656
Red Buffer (10X)27
EcoRI0.250.875
PstI0.250.875
  • Amount per tube multiplied by 3.5

Add 18µL master mix to each plasmid DNA sample
Pipetting Scheme for Unrestricted reactions:

IngredientVolume/tube (µL)Total Volume*
MilliQ H2O1656
Red Buffer (10X)27
  • Amount per tube multiplied by 3.5

Add 18µL master mix to each plasmid DNA sample
Buffer Control will be 18µL MilliQ H2O + 2µL 10x Red Buffer.
Place in 37oC water bath at 12:37pm and removed at 1:55pm for approximately 1 hour incubation.
Analyze samples on a 1% agarose gel (small gel apparatus).
Add 3.3µL of 6x DNA loading dye to each reaction mixture and load.

Lane</td>Sample</td>Volume Loaded (µL)</td>
11 kb Ladder4
2Restricted sRBS (E10)10
3sRBS (E10)10
4Restricted sRBS (D5)10
5sRBS (D5)10
6Restricted sRBS-Lumazine-dT10
7sRBS-Lumazine-dT10
8Red Buffer Control10

Ran gel at 100V for 75 minutes (Start-2:30pm; End-3:45pm)
Stained in ethidium bromide for 10 minutes

Results:

100515JV.jpg

There is plasmid DNA in each sample which, when cut with both the prefix and suffix enzyme, yields a band approximately 2000bp (size of pSB1A3 is 2157bp).

May 17/2010

(in the lab: JV, AV)
Make agar plates with 100µg/mL of ampicillin
Make 5 x 5mL sterile liquid SOC broth
Make 13 x 5mL sterile liquid LB broth

May 17/2010 Evening

(in the lab: TF, AS)
Objective: To grow cells for future use
Streaked plates from glycerol stocks of the following:
iGEM 2007 -80oC Freezer Box:

ConstructCell typeLocation of cells
xylEDH5αC4
xylEBL21(DE3)B4
C-term BbaDH5αH4
C-term BbaDH5αI4
C-term BbaDH5αJ4
Mr. Gene mms6DH5αA6
Mr. Gene mms6DH5αB6

iGEM 2010 -80oC Freezer Box:

ConstructCell typeLocation of cells
pLacIDH5αB1
dTBL21(DE3)D1

Incubated at 37oC, beginning at 20h00 (8:00pm)

Made liquid cultures from cells taken from transformation plates (grown on May 13/2010):

  • sRBS (D5)
  • sRBS(E10)
  • Lumazine-dT (1)
  • pBad-TetR

Incubated at 37oC, beginning at 20h30 (8:30pm); shaking at 300RPM
Results:
All streak plates (with cells from glycerol stocks) grew.
Only sRBS (D5) and pBad-TetR cells (from transformation plates) grew.

May 18/2010

(in the lab: JV, AV, HB)
NOTE: Cells from liquid cultures grown last night (May 17/2010) were made into glycerol stocks and placed into the working glycerol stock box as follows:

  • pBAD-TetR - E5
  • pBAD-TetR - E6
  • sRBS (D5) - E7
  • sRBS (D5) - E8

Objective: To isolate plasmid DNA of pBad-TetR and sRBS (D5) and cut with restriction enzymes.

Method:
Use boiling lysis miniprep to prepare plasmid DNA. Digest sRBS with PstI only; digest pBAD-TetR with SpeI (old and new) and PstI.

Reaction conditions for PstI using Orange Buffer.

IngredientVolume/tube (µL)
Milli-Q H2O15.75
Orange Buffer2
Plasmid DNA2
PstI0.25

Reaction conditions for PstI using Tango Buffer.

IngredientVolume/tube (µL)
Milli-Q H2O15.75
Tango Buffer2
Plasmid DNA2
PstI0.25 & 0.25

Objective: To Restrict pLacI (D2) and sRBS-Lumazine Synthaze-dT (A2) and ligate them together

Method:
Reaction conditions for the Plasmid DNA control.

IngredientVolume/tube (µL)
Milli-Q H2O16
Tango Buffer2
Plasmid DNA (pLacI or sRBS-Lum-dT)2

Reaction conditions for XbalI, and PstI using Tango Buffer.

IngredientVolume/tube (µL)
Milli-Q H2O15.5
Tango Buffer2
Plasmid DNA (sRBS-Lum-dT)2
XbaI & PstI0.25 & 0.25

Reaction conditions for SpeI, and PstI using Tango Buffer.

IngredientVolume/tube (µL)
Milli-Q H2O15.5
Tango Buffer2
Plasmid DNA (sRBS-Lum-dT)2
SpeI & PstI0.25 & 0.25

Buffer control contains: 18µL Milli-Q H2O, and 2µL Tango Buffer.

Incubated at 37oC from 1:30pm to 2:55pm.
Analyze results on 1% Agarose gel (in 1x TAE); NOTE: Sample mixed with loading dye prior to loading onto gel.

LaneSampleVolume Sample (µL)Volume Dye (µL)
1pBad-TetR Restricted with old SpeI5.01.0
2pBad-TetR Restricted with new SpeI5.01.0
3pBad-TetR Unestricted (Tango Buffer)5.01.0
4pBad-TetR Restricted with PstI5.01.0
5pBad-TetR Unrestricted (Orange Buffer)5.01.0
6sRBS Restricted with PstI5.01.0
7sRBS Unrestricted5.01.0
8Buffer Control (Tango)5.01.0
9Buffer Control (Tango)5.01.0
10Empty5.01.0
11Empty5.01.0
12Empty5.01.0
13Empty5.01.0
14Empty5.01.0
15Empty5.01.0
16sRBS-Lum-dT Unrestricted5.01.0
17sRBS-Lum-dT Restricted with XbaI/PstI5.01.0
18pLacI Unrestricted5.01.0
19pLacI Restricted with SpeI/PstI5.01.0
201kb Ladder2.50.5

Ran gel at 100V for 90 minutes.
Results:

100518JV1.JPG

Objective: Make liquid cultures of streak plates (made May 17/2010) for plasmid mini-preps.
Method: Added 5µL of 100mg/mL ampicillin to 5mL of LB liquid broth to give a final concentration of 100µg/mL ampicillin.
Picked cells from single colonies and inoculated into 5mL LB (Amp+) media of the following constructs:

  • C-term BBa (I4-2007 Box)
  • C-term BBa (J4-2007 Box)
  • C-term BBa (H4-2007 Box)
  • dT (D1-2010 Box)
  • pLacI (B1-2010 Box)
  • mr. Gene mms6 (A6-2007 Box)
  • mr. Gene mms6 (B6-2007 Box)
  • xylE (B4-2007 Box)
  • xylE (C4-2007 Box)

May 18/2010 Evening

(in the lab: KG)
Objective: Restrict pLacI, sRNS, sRBS-Lumazine Synthase-dt out of plasmid then ligase pLacI and sRBS also pLacI sRBS-Lumazine Synthase-dt.

Method:
Restriction Digestion
Tube 1 contains:

IngredientVolume/tube (µL)
Milli-Q H2O31.50
Red Buffer4
Plasmid DNA (pLacI)4
EcoRI & SpeI0.50 & 0.50

Tube 2 contains:

IngredientVolume/tube (µL)
Milli-Q H2O15.75
Tango Buffer2
Plasmid DNA (sRBS)2
XbaI & PstI0.25 & 0.25

Tube 3 contains:

IngredientVolume/tube (µL)
Milli-Q H2O15.75
Red Buffer2
Plasmid DNA (pLacI)2
XbaI & PstI0.25 & 0.25

Incubated at 37oC for 1 hour starting at 7:00pm.
After incubation tubes 1,2, and 3 were placed on a heating bloack for 10 minutes at 65oC (Start-8:08pm and 8:18pm). Also heated samples from JV restriction with pLacI only cut at the suffix.

Ligation
Tubes 4,5, and 6

IngredientVolume/tube (µL)
Ligation Buffer2
Milli-Q H2O17
T4 DNA Ligase1

Tube 4 contains:

sRBS from Restriction</td><td></table>
Tube 5 contains:
IngredientVolume/tube (µL)
Milli-Q H2O2.75
Ligation Buffer2
T4 DNA Ligase0.25
PlacI from restriction(double cut)7.5
sRBS-Lumazine Synthase-dt
IngredientVolume/tube (µL)
Milli-Q H2O2.75
Ligation Buffer2
T4 DNA Ligase0.25
PlacI from restriction(double cut)7.5

Tube 6 contains:

sRBS-Lumazine Synthase-dt
IngredientVolume/tube (µL)
Milli-Q H2O2.75
Ligation Buffer2
T4 DNA Ligase0.25
PlacI from restriction(single cut)7.5

Begin room temperature incubation at 8:25pm.

May 19/2010

(in the lab:JV)

Objective:Isolate and restrict plasmid DNA from liquid cultures started May 18, 2010.

Method:
Use boiling lysis miniprep to prepare plasmid DNA.

  • C-term BBa (I4-2007 Box)
  • C-term BBa (J4-2007 Box)
  • C-term BBa (H4-2007 Box)
  • dT (D1-2010 Box)
  • pLacI (B1-2010 Box)
  • mr. Gene mms6 (A6-2007 Box)
  • mr. Gene mms6 (B6-2007 Box)
  • xylE (B4-2007 Box)
  • xylE (C4-2007 Box)

Restriction Digestion of prepared plasmid DNA
Master Mix 1:

IngredientVolume/tube (µL)(Volume/tube(µL)) X 10
Milli-Q H2O15.75157.5
Orange Buffer220
EcoRI0.252.5

Master Mix 2:

IngredientVolume/tube (µL)(Volume/tube(µL)) X 10
Milli-Q H2O16160
Orange Buffer220

Add 2(µL) of plasmid DNA to 18(µL) of master mix to create restriction digestion reaction.
Buffer control contains 2(µL) of orange buffer and 18(µL) Milli-Q H2O.
Reactions ran for 1 hour at 37oC.

May 20/2010

(in the lab:JV, AV)

Objective:Check plasmid DNA and restriction digest reactions prepared May 19,2010.

Method:
Analyzed restriction digests on a 1% agarose gel (large gel apparatus)
Added 1µL of 6x DNA loading dye to 5µL of sample
Added 1µL of 6x DNA loading dye to 4µL of Milli-Q H2O and 1µL of 1kb DNA mass ladder.
Loaded samples as follows:

LaneSampleVolume Loaded (µL)
11 kb Ladder5
2Buffer Control5
3pLacI (B1) restricted5
4pLacI (B1) unrestricted5
5dT (B1) restricted5
6dT (B1) unrestricted5
7mms6 (A6) restricted5
8mms6 (A6) unrestricted5
9mms6 (B6) restricted5
10mms6 (B6) unrestricted5
11xylE (B4) restricted5
12xylE (B4) unrestricted5
13xylE (C4) restricted5
14xylE (C4) unrestricted5
15C-term (H4) restricted5
16C-term (H4) unrestricted5
17C-term (J4) restricted5
18C-term (J4) unrestricted5
19C-term (I4) restricted5
20C-term (I4) unrestricted5

Ran gel at 100V for 2 hours
Results:

100520AV.JV-Plasmid Check.JPG

May 20/2010 Evening

(in the lab:JS, AS)

Objective: Re-run the gel from earlier in the day with a higher DNA concentration

Method:
Analyzed restriction digests on a 1% agarose gel (large gel apparatus)
Added 2µL of 6x DNA loading dye to 10µL of sample
Added 2µL of 6x DNA loading dye to 8µL of Milli-Q H2O and 2µL of 1kb DNA mass ladder.
Loaded samples as follows:

LaneSampleVolume Loaded (µL)
11 kb Ladder5
2pLacI (B1) unrestricted10
3pLacI (B1) restricted10
4dT (B1) unrestricted10
5dT (B1) restricted10
6mms6 (A6) unrestricted10
7mms6 (A6) restricted10
8mms6 (B6) unrestricted10
9mms6 (B6) restricted10
10xylE (B4) unrestricted10
11xylE (B4) restricted10
12xylE (C4) unrestricted10
13xylE (C4) restricted10
14C-term (H4) unrestricted10
15C-term (H4) restricted10
16C-term (I4) unrestricted10
17C-term (I4) restricted10
18C-term (J4) unrestricted10
19C-term (J4) restricted10
20Buffer Control10

Ran gel for 80 minutes at 100V.
Results:

100520JS1.JPG

May 25/2010

(in the lab:JV, HB, AV)

Objective: Transform the following ligated parts:

  • pLacI-sRBS (single cut from May 18,2010)
  • pLacI-sRBS (double cut from May 18,2010)
  • pLacI-sRBS-lumazine synthase-dt (from May 18, 2010)

Method:
Used Competent Cell Transformation to prepare plasmid DNA.
Results:
No colonies grew. No colonies on positive control plate. Something amiss here.
NOTE (June 1/2010; AS): Likely that the problem here was the method used to heat shock the cells. Cells were heat shocked in a heat plate, with incomplete contact of the heating surface, causing inefficient transfer of heat to cells for shock and movement of DNA into competent cells.

May 25/2010 Evening

(in the lab: AV, KG)

Objective:Restriction of dt, xylE, and mms6. To ligate xylE with dt and mms6 with dt.

Method:

  • Cut dt with XbaI and PstI (Buffer: Tango)
  • Cut xylE with EcoRI and SpeI (Buffer: Red)
  • Cut mms6 with EcoRI and SpeI (Buffer: Red)

Restriction of above plasmids.

Tube 1:

IngredientVolume/tube (µL)(Volume/tube(µL)) X 4
Milli-Q H2O15.5155
Tango28
plasmid (dt)28
XbaI & PstI0.25 & 0.250.5 & 0.5

Tube 2:

IngredientVolume/tube (µL)
Milli-Q H2O15.5
Red2
plasmid (mms6(A3))2
EcoRI & SpeI0.25 & 0.25

Tube 3:

IngredientVolume/tube (µL)
Milli-Q H2O15.5
Red2
plasmid (mms6(B3))2
EcoRI & SpeI0.25 & 0.25

Tube 4:

IngredientVolume/tube (µL)
Milli-Q H2O15.5
Red2
plasmid (xylE(B4))2
EcoRI & SpeI0.25 & 0.25

Tube 5:

IngredientVolume/tube (µL)
Milli-Q H2O15.5
Red2
plasmid (xylE (C4)2
EcoRI & SpeI0.25 & 0.25

Incubated on heat block set at 37oC between 7:19-8:19pm

Objective: Ligate mms6 to dt, and xylE to dt (JV, AV, HB, KG, AS)

Method:
Tube 1:

IngredientVolume/tube (µL)
Milli-Q H2O2.75
Ligation Buffer2
T4 DNA Ligase0.25
mms6 (B6)7.5
dt (B1)7.5

Tube 2:

IngredientVolume/tube (µL)
Milli-Q H2O2.75
Ligation Buffer2
T4 DNA Ligase0.25
mms6 (A6)7.5
dt (B1)7.5

Tube 3:

IngredientVolume/tube (µL)
Milli-Q H2O2.75
Ligation Buffer2
T4 DNA Ligase0.25
xylE (B4)7.5
dt (B1)7.5

Tube 4:

IngredientVolume/tube (µL)
Milli-Q H2O2.75
Ligation Buffer2
T4 DNA Ligase0.25
xylE (C4)7.5
dt (B1)7.5

  • T4 DNA ligase added to ligations at 8:37pm.
  • Incubated reactions overnight at room temperature
  • Inactived by heating for 10 minutes at 80oC at 10:00am

Analyze restrictions and ligations by electrophoresis on a 1% agarose gel, stained with ethidium bromide. Load order is as follow:

LaneSampleVolume Rxn (µL)Volume Dye (µL)
1Restricted mms6 (B6)1010
2Unrestricted mms6 (B6)1010
3Restricted mms6 (A6)1010
4Unrestricted mms6 (A6)1010
5mms6 (B6)-dT ligation1010
6mms6 (A6)-dT Ligation1010
7Restricted dT (B1)1010
8Unrestricted dT (B1)1010
9Restricted xylE (C4)1010
10Unrestricted xylE (C4)1010
11Restricted xylE (B4)1010
12Unrestricted xylE (B4)1010
13xylE (C4)-dT Ligation1010
14xylE (B4)-dT Ligation1010
151kb Ladder2 (+8water)2
16Empty
17Empty
18Empty
19Empty
20Empty

Gel was run at 100V for 2 hours
Results:

100525 JV XYLE MMS6 Ligation.jpg

Objective:Grow overnight cultures of cells for pDNA mini-prep tomorrow.
Method:
DH5α cells from -80oC freezer containing the below genes were inoculated into 5mL LB liquid broth containing 100µg/mL of ampicillin and subsequently incubated at 37oC with shaking (at 300RPM) overnight (beginning at 7:30pm)
Cells with the following gene products were retrieved from the -80oC freezer:

Common NameBoxLocationResult
Fusion CEYFP2007H5,I5,J5All grew
CEYFP2007G6,H6All grew
NEYFP2007I6,J6All grew
Arg N-term2007I7,J7None grew
Arg C-term2007J8All grew
ECFP2009A3,B3,C3All grew
EYFP2009A6,B6,C6All grew
TetR Inverter2009A9,B9None grew
pBad-TetR2007/2009G8/C9All grew

Removed at 9:30pm

May 26/2010

(In the lab: JV)
Objective: Purify plasmid DNA (via mini-prep) of the cells grown last night.
Method: Performed boiling lysis miniprep as described in protocols section on cells containing the following genes:

  • Fusion CEYFP (H5 - 2007)
  • Fusion CEYFP (J5 - 2007)
  • NEYFP (J6 - 2007)
  • NEYFP (I6 - 2007)
  • Fusion CEYFP (I5 - 2007)
  • CEYFP (G6 - 2007)
  • pBad-TetR (G8 - 2007)
  • CEYFP (H6 - 2007)
  • EYFP (A6 - 2009)
  • ECFP (A3 - 2009)
  • pBad-TetR (C9 - 2009)
  • ECFP (C3 - 2009)
  • EYFP (B6 - 2009)
  • EYFP (C6 - 2009)
  • ECFP (B3 - 2009)

Results: Will restrict and run agarose gels tomorrow.

May 27/2010

(In the lab: JV, AV)

Objective:Restrict and run agarose gels of plasmids 'minipreped' yesterday from the 2007 and 2008 glycerol stock boxes.

Method:
Master Mix for Restriction Reactions:

IngredientVolume/tube (µL)(Volume/tube(µL)) X 16
Milli-Q H2O15.75252
Orange232
EcoRI0.254

Master Mix for unrestricted DNA:

IngredientVolume/tube (µL)(Volume/tube(µL)) X 16
Milli-Q H2O16256
Orange232

18µL of restriction master mix was added to 2(µL) of each plasmid.
18µL of unrestricted master mix was added to 2(µL) of each plasmid.
18µL of Milli-Q H2O was added to 2(µL) of orange buffer as a buffer control.
Reactions were carried out for 1 hour at 37oC.
Analyzed on a 1% agarose gel.

Gel 1

LaneSampleVolume Rxn (µL)Volume Dye (µL)
11kb Ladder†22
2Buffer Control1010
3CEYFP (H6) Restricted1010
4CEYFP (H6) Unrestricted1010
5CEYFP (G6) Restricted1010
6CEYFP (G6) Unrestricted1010
7EYFP (C6) Restricted1010
8EYFP (C6) Unrestricted1010
9Fusion CEYFP (I5) Restricted1010
10Fusion CEYFP (I5) Unrestricted1010
11EYFP (C3) Restricted1010
12EYFP (C3) Unrestricted1010
13pBad-TetR (C9) Restricted1010
14pBad-TetR (C9) Unrestricted1010
15ECFP (A3) Restricted1010
16ECFP (A3) Unrestricted1010
17Fusion CEYFP (H5) Restricted1010
18Fusion CEYFP (H5) unrestricted1010
19pBad-TetR (G8) Restricted1010
20pBad-TetR (G8) unrestricted1010

† Also added 8µL of water to this mix

Gel 2

LaneSampleVolume Rxn (µL)Volume Dye (µL)
11kb Ladder†22
2Restricted Fusion CEYFP (J5)1010
3Unrestriced Fusion CEYFP1010
4Restricted ECFP (B3)1010
5Unrestricted ECFP (B3)1010
6Restricted EYFP (B6)1010
7Unrestricted EYFP (B6)1010
8Restricted NEYFP (I6)1010
9Unrestricted NEYFP (I6)1010
10Restricted EYFP (A6)1010
11Unrestricted EYFP (A6)1010
12Restricted EYFP (J6)1010
13Unrestricted EYFP (J6)1010

† Also added 8µL of water to this mix

Ran both gels at 100V for 2 hours.
Results:
Gel 1

100527 AV Plasmid Test 1.jpg

Gel 2

100527 JV Plasmid Test 1.jpg

Conclusion: To be concluded....

Objective: Transform DH5α cells with plasmids containing ligation products from May 25/2010.
Method: Transform, using the competent cell transformation protocol, the ligation products with the most intense banding from each of the following ligation reactions:

  • mms6 + dT (B6 + A6)
  • xylE + dT (B4 + C4)

Incubate at 37oC overnight.
Results:
No colonies grew, even on positive control plate.

May 31/2010

Objective: Transform part BBa_J33204 (Ribosomal Binding Site + xylE gene) into DH5α cells.
Method:
Obtained part BBa_J33204 from 2010 iGEM Spring Distribution Kit Plate 1, well 7P.
Transformed using competent cell transformation protocol, along with negative control (milliQ H2O water) and positive control (pUC19 plasmid). Plated 100µL and 50µL on separate pre-warmed LB agar plates containing 100µg/mL ampicillin.
Added 250µL of SOC media and incubated for 90 minutes at 37o.
Results:
Obtained the following number of colonies on each plate:

  • BBa_J33204 50µL - 13 colonies
  • BBa_J33204 100µL - 18 colonies
  • pUC19 - 9 colonies

Other lab activities:

  • Inoculated 5mL of LB liquid broth (Amp+ with cells containing Bba_J33204 picked from colony off transformation plate and incubated at 37oC with shaking overnight.

AV quantified each pDNA stock in the working plasmids box by measuring the A260 of a 1:100 dilution of the pDNA samples in a UV cuvette. Results are posted on the working plasmids page.

May 31/2010 - Evening

Objective: Transform recent ligation reactions that didn't work the first time around.
Method: Use competent cell transformation protocol to transform the following ligation products:

  • pLacI + sRBS
  • pLacI + sRBS-Lum-dT (times 2)
  • mms6 (B6) + dT
  • mms6 (A6) + dT
  • xylE (C4) + dT
  • xylE (B4) + dT

Plated all cells; spun down media so cells were pelleted and removed 200uL of cell-free media. Re-suspended pelleted cells in remaining media, and plated onto ampicillin plates.
Results:
No growth on plates EXCEPT:

  • Positive control (pUC19) - 9 colonies
  • mms6 (B6) + dT - 1 colonies


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