Team 1

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Procedure:
Procedure:
Legend: Black: Basic Summary
Legend: Black: Basic Summary

Latest revision as of 20:12, 27 October 2006

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Procedure: Legend: Black: Basic Summary Red !!!DET!!!, details still missing, Red anything: Detail not 100% CONFIRMED, NEED TO CHECK OTHER PEOPLE’S Lab Notebooks.

June 21 Wednesday:

  • Arrival of DNA from Spain (send by the Estaban Veiga, Víctor de Lorenzo, and Luis Angel Fernández team), 2 plasmids, one (!!!DET!!!) Jun-Beta, and the other (!!!DET!!!) Fos-Beta. They arrived in the form of dried out (!!!DET!!!) bacteria that were placed in the fridge at 4OC. These will serve as the Vectors. These are under chloramphenicol resistance


June 27 Tuesday:

  • Arrival of a plasmid from Michigan that contains both inserts Fos-YFPC and Jun-YFPN on pDS-BiFC plasmid. This arrived in the form of wet dilute DNA, temporary storage 4OC: amp resistance.
  • Transformation of both Michigan Plasmids into Top Ten chemically competent E.coli cells.

One test tube of Top 10 homemade competent cells taken from –80 to ice bucket, thawed at room temp. (One since both inserts in same plasmid)

  • Melted SOC medium at 37OC.

o Added 3μL DNA into bacterial tube and placed in ice incubation for 30 minutes. o Heat shock 2 minutes, added 250μL SOC medium, ice 1 minute, 1h in 37OC shaker incubator. o Spread on two Amp plates150μL and 50μL, placed at 37OC incubation room overnight.


June 28 Wednesday:

  • 10:07 am: Transformation Results: Many tiny colonies on 150μL plate, Larger colonies in 50μL plate. They were placed in fridge.
  • 2ml seedings of Fos-Beta, and Jun-Beta were performed.

o 11:08AM: Tubes numbered in the following manner:

1. Spanish Jun June 28 (for Fos-Beta)

2. Spanish Fos June 28 (for Jun-Beta)

5. Control cm (meaning chlorampenicol)

6. Control no amp cm.

o 11:20 am: 10ml pipette used to add 1ml 2X LB to each tube.

o 11:24 am: 10ml pipette used to add 1ml dH2O to each tube.

o 11:31 am: 7μL of 30ng/μl chloramphenicol added to tubes 1, 2, and 5. (Note: This was actually an overload of chloramphenicol as a result of mistake, the proper amount of chroramphenicol would have been 2 μL, and nevertheless growth was positive regardless).

o 11:40 am: Fos-YFPC seeded into tube 1 and Jun-YFPN (plasmids) was seeded to tube 2 by picking straight from tube of cells from Spain with a sterile micropipette tip and ejecting into tubes 1 and 2.

o 11:45 am: Incubation in 37OC shaker incubator begins.

  • 20:00 pm: 500μL of each seedings were seeded into a larger 25ml midiprep seeding in large Erlenmeyer flask. This seeding consisted of 12.5mL each sterile dH2O and sterile 2X LB and 25μL chloramphenicol. Covered with aluminium foil and placed in 37OC shaker incubator overnight.


June 29 Thursday:

  • 9:56 am: seedings grew very well, therefore midiprep procedure (using Quiagen midiprep kit) began.
  • 11:11 am: 4 large centrifuge tubes were washed with Javex Bleach, rinsed 10 times with dH2O, and labelled 1 and 2. 1 was used for Fos-Beta and 2 for Jun-Beta. The othe 2 tubeslabeled 3 and 4 were reserve.
  • 11:23 am: 25mL of Fos overnight culture added to 1 and 25mL Jun culture added to 2 using 10mL sterile pipettes.
  • Centrifugation of both tubes at 20, 000 g’s for 15 minutes at 25OC done.
  • 13:42 pm: Supernatant poured off, re-suspended in 4ml P1 Quiagen buffer and vortexed.
  • 14:00 pm: 4mL Quiagen buffer P2 added (note: tube 1 did not turn clear, nevertheless procedure continued), tubes inverted 6 times, waitd roughly 3-4 minutes and added buffer P3, inverted 6 times. Tubes placed in ice for 17 mintes.
  • 14:24-14:54 pm: Tubes centrifuged 30 minutes, 4OC, 20, 000g’s.
  • Meanwhile, equilibrated 2 QUIAGEN columns with water.
  • 15:00 pm: Supernatant from centrifugation added to the QUIAGEN columns (first removing the scum).
  • 15:32 pm: First wash by adding 10mL Quiagen buffer QC to each column.
  • 15:50 pm: Second wash, 10mL QC. Assigned for elusion, centrifuge tube 2 as Fos and 4 as Jun.
  • 16:20 pm: Placed QUIAGEN columns above centrifuge tubes 3 and 4 and eluted by adding 5 ml buffer QF using 10mL sterile pipettes.
  • By 17:55 pm: After elusion, centrifuged 30min, 4OC, 15000 g’s. Than supernatant removed and solution placed in 1ml ethanol (dissolved by pipetting up and down), contents of tube transferred to smaller 1500μL centrifuge tube. Than 5 min centrifugation at 14000 rpm on able top centrifuge was performed, followed by removal of supernatant, addition of another 1mL ethanol, another 5 min max speed centrifugation. Supernatant was poured off, air dried and 50 mL of sterile dH2O was added. (Note: Later on the 16th of August, this was diluted 4 fold Fos to 45μL DNA added 180μL dH2O, Jun 140μL dH2O added to 35μL DNA. Also please refer to Friday July 28th for determination of midiprep concentration).


July 1 Monday:

  • 11:52 am: There was a need to screen the midipreps to confirm this is the right DNA.The program GCK found that:

1. Fos: Uncut Plasmid: 5362bp; BamHI cut: 1442bp and 3920bp

2. Jun: Sizes would be almost exactly the same.

  • 13:07 pm: The following screening digest was set up (2 for each Jun and Fos):

1. 10.0μL dH2O

2. 1.5μL NEB (New England Biolabs) Buffer 2

3. 1.0μL midiprep DNA (Jun or Fos)

4. 1.5μL 10X BSA (this one with lower accuracy)

5. 1.0μL NEB BamHI

  • After completion 1h incubation in 37OC water bath begins.
  • Meanwhile a 0.7% agarose Gel was poured. First addition of 50ml TAE buffer in 0.35g agarose in Erlenmeyer flask, than microwaving for about 1 minute, cooling to below 60OC adding 5μL Ethidium Bromide and Pouring onto a tray.
  • 15:45 pm: loaded a gel in the following manner

1. 12μL DNA ladder

2. Fos-Beta BamHI digest 18μL (15μL digest, 3μL loading dye)

3. Fos-Beta undigested DNA 18μL (1μL DNA, 14μL dH2O, 3μL loading dye)

4. Fos-Beta undigested DNA 18μL (2μL DNA, 13μL dH2O, 3μL loading dye)

5. Jun-Beta BamHI digest 18μL (15μL digest, 3μL loading dye)

6. Jub-Beta undigested DNA 18μL (1μL DNA, 14μL dH2O, 3μL loading dye)

7. Jun-Beta undigested DNA 18μL (2μL DNA, 13μL dH2O, 3μL loading dye)


  • The results show that in case of Jun-Beta the right bands are seen for the digest, but in the case of Fos-Beta the 3920bp band is seen but not the 1442bp band. In addition the amount of DNA added seams to be high (i.e. midiprep highly concentrated).


July 6 Thursday:

  • 11:10 am: More Confirmation was required for the Fos-Beta midiprep, therefore another BamHI digest was performed:

1. 10μL dH2O

2. 1.0μL DNA

3. 1.5μL NEB Buffer 2

4. 1.5μL NEB BSA

5. 1.0μL NEB BamHI

  • 13:00 pm: 2 gels were poured on two trays using 100mL TAE, 0.70g agarose and 100μL EtBr.
  • 13:30 pm: A gel was poured in the following manner:

1. DNA ladder (10μL)

2. DNA ladder (5μL, IRRELEVANT, experimental purpose to see f 5μL enough to see)

3. Fos Beta (15μL digest, 3μL loading dye)


  • The bands are in the right place. The upper band corresponds to the 4000bp band in ladder (makes sense since it’s 3920) and lower band corresponds to 1500bp band and it should be 1442bp.
  • Also this week PCR primers arrived: GUYS DO YOU KNOW WHEN THEY ARRIVED, WHO DISSSOLVED THEM, AND WHAT THEY ARE???

Week of July 10th 2006: Julia Please Specify when you seeded and miniprepped and digested to confirm pDS-BiFC-JunYFPN-FosYFPC, thanks


July 18 Tuesday:

  • 11:26 am: Two 0.8% gels were made using 0.8g agarose, 100mL TAE, microwaved and cooled to 50OC, then 4.25μL EtBr added.
  • 12:03 pm: The gel was loaded with 8μL ladder in first lane and 8 consecutive overnight digests of Jun-YFPN-Fos-YFPC on pDS-BiFC plasmid (for insert). Gel ran 45 minutes.


  • From this result, one can see that the first digest has the right plasmid since the bands are in the right place. Therefore this is the plasmid that is utilized for the PCR.
  • 15:42 pm: For PCR, a 50X dilution (since this is a miniprep) had to be made of miniprep 1 on lane 2 of Figure 3 Gel. Mixing 2μL miniprep DNA with 98μL of PCR water did this.


July 27 Thursday

  • 11:50 am: Four PCR reactions were performed today, two of each FosYFPC and JunYFPN. All the substances were added using sterile filter pipetter tips and only sterilized, distilled PCR water was used. Small PCR tubes were used. The following order of addition was applied.

1. 80μL PCR water

2. 10μL PCR buffer

3. 4μL dNTP’s

4. 2μL forward primer

5. 2μL back primer

6. 1μL BSA

7. 1μL Taq polymerase (Note: this is added using freezer box, only taken once necessary)

8. 1μL pDiFC (Note: Same template used for both PCR reactions)

9. The tubes were inserted in ?42? cycle PCR machine for 3 hours.

  • 12:25 pm: A 1.2% gel had to be poured (due to our PCR products being 281bp for YFPC (fos) and 488bp for YFPN (Jun). This was done by 1.2g agarose
  • Than a PCR purification procedure was performed (Alex and Belinda). A small amount of this was ran on a gel for the purpose of screening.
  • The Gel Was loaded in the following manner:

1. 6μL Gel Pilot 100bp+ladder

2. bad well

3. 5 μL DNA ladder

4. YFPN (for Jun first PCR reaction)

5. YFPN (For Jun, second PCR reaction)

6. YFPC (For Fos, first PCR reaction)

7. YFPC (For Fos, second PCR rection)

  • The Gel results were very good, the PCR and the Purification worked very well.


July 28 Friday

  • The concentration of the PCR product was determined by comparing to the DNA ladder in the following manner.

124ng x1μL ladder x1 x5μL ladder used =24.5ng/μL 1μgDNA 1μL ladder 10 dil factor 2.5μL DNAused

  • The values are about the same for all PCR reactions and we have 100μL of each. From this can be seen that there is plenty of PCR product for insert.
  • Estimation of concentration of Jun-Beta and Fos-Beta midiprep vectors using spectrophotometric techniques. A small amount of the midiprep was diluted 1000 fold
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