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From 2010.igem.org

Tuesday 9/7/2010

 

Results from 9/3/2010:

No growth on the plate from 9/3/2010. BL21 cells can not grow by themselves on plates with antibiotic. The vectors we normally add contain antibiotic resistance genes to select for the colonies with our genes, BL21 by themselves don’t contain these resistance genes.  

 

Note that the “Experimental things” notes on 9/3/2010 is NOT experimental procedure. Please write completely and explicitly all steps.. even when just plating cells. For example: How much of the BL21 cells were used? Were they frozen? Were they thawed? If so, how were they thawed? Water bath? Room temp? Pocket? Was anything else added? What temperature were they kept at? Where were plates kept and for how long? What was the plate labelled? What to do with them next? etc.

 

We need experimental procedure and results at a MINIMUM. We should also have purpose, summaries of results, implications, hypotheses and even background information (example - why use BL21 instead of Novablue?).  This should keep everybody up-to-date and help with coordination so people aren’t backtracking to figure out what people did and why.

-Christina and Rob

 

 

We have the iGem plates containing dry hybB, we have regular ompA, and we now have dry Aox genes. We want to grow up many copies of these, so we need to transform and grow cultures of them.

 

We began by reconstituting (adding water) to all the dry DNA:

50 uL for both AOX1a and AOX1b tubes

10 uL to the hybB well (iGem Plate 2, well 7M) [observed an orange color solution]

10 uL to the pSB1A3 backbone vector (iGem Plate 1, well 1C) [observed an orange color solution]

 

5 transformations were performed to grow future stocks:

1. AOX1a in Novablue

2. AOX1b in Novablue

3. ompA in Novablue

4. hybB in Novablue

5. pSB1A3 in Novablue

 

Heat shock transformation of the plasmids into our bacteria

10 սL Nova Blue cells + 5 սL of each target plasmid

See Protocols page for Heat Shock Transformation

 

 

It was noted that several plates had heavy condensation- not sure if that might affect colony growth/development, so all were placed in the cell culture hood after plating to dry off the plates.

The resulting plates (10 plates ttl, 2 for each transformation) were labeled and placed inside the 37C incubator in the iGem lab.

 

For next time:

Colonies should be selected for each of the 5 transformations, cell cultures for each should be grown in LB media, and incubated overnight.

 

9/8/2010

Results

1. Plates from the transformations on 9/7/2010 all had growth. The aoxa/b plates had many colonies (similar to a “lawn” of growth). Ryan suggested that for plasmids, we reduce the amount of plasmid we add to the cells during transformation (from 5uL to 1 uL). Transformations with plasmids require only 25-50 ng of DNA.
2. Replate using triple smear technique (see below) to get distinct colonies.

 

Goals:

Pick 1 colony from each of the plates from the transformations done 9/7/2010, inoculate a 5 ml of LB+Carb in Falcon round bottom tube. Grow these overnight @37c. Then on 9/9/2010, we can do 5 minipreps and 5 cryostocks (a.k.a DMSO stocks, where we freeze cells @-80c) on the starter cultures. The end result will be isolated plasmids of hyBb, AOXa/b, pSB1A3, and ompA.

Actions:

1. I moved the plates from 9/7/2010 from 37C into the 4C refrigerator. Plates last longer in the refrigerator- at 37, they will overgrow!
2. I created liquid cultures (per protocol listed below on 9/8/2010) 10am-11am for each of the transformants. They are at RT now and will place them in incubator around 5pm.

Liquid Cultures

3. +5mL LB into 5 50mL eppendorfs. (changed to falcon round bottom tubes appropriate for incubator/shaker).

1. +50 uL 1000x Carb (antibiotic)
2. Using a toothpick, gently scrape off and pick 1 discrete colony from a plate and put it into the falcon round bottom tube containing the LB+Carb.
3. Put in incubator shaker 37C overnight (time on: 3:30 pm 9/8/10).

 

For Future/tomorrow:

We are preparing liquid culture since we want to perform minipreps on the cells, isolate plasmids, and also create cryostocks of the transformed cells future use. Perform all of these tomorrow!

Scott

 

2pm: Gita and Debika

Aliquoting the primers: all need to  b r PCR.

1. centrifuge the tubes for 1 minute at 2000 rpm to get all of DNA at the bottom of the tube.

2. add autoclaved water to dilute. ex.  36.2 nM --> add 362 microliters of water

3. vortex, wait for 20 min, revortex

4. store in freezer at -20 C

 

The plates grown up on 9/7/2010 do not contain discrete colonies, as the plates were wet when the cells went on.  In order to ameliorate this problem, we will do a triple smear and replate so we can obtain distinct colonies.

 

Richard showed us the triple smearing technique. Christina placed all plates made yesterday under the hood for them to dry up. After an hour, she will check back on them and perform triple smearing.

Triple Smearing Technique:

 

All of the plates that we have currently in the 4C fridge need to be dried in the hood before use... When we originally made them, they weren’t allowed to cool long enough (should have been about 1 hour) before putting them in the fridge.  As a result, the plates are pretty wet. Dry them first. (Note: 9/10/10 - all plates have been dried).

 

Replating using smearing technique. Plates are labelled with: ‘plasmid’:novablue; 9/8/2010, iGEM.

Placed in the 37C incubator (in iGEM lab) overnight.

Note: plates discarded on 9/9/2010.

 

9/9/2010

Results from 9/8/2010:

A) Triple smear:

Too many cells. Need to retry this technique. Plates thrown out.

 

B) Starter colonies:

Successful growth of starter colonies prepared on 9/8/2010.

 

Procedure:

A) Triple smear replating (hybB, ompA, Aox1a, Aox1b, pSB1A3):

1. Dry all plates in hood.

2. Try the triple smear replating technique again. Replate as on 9/8/2010 again using triple smear technique - too many cells were gathered on 9/8/2010, those plates were thrown out.  Make sure to obtain a very small amount of cells.

 

B) Remake some starter colonies using distinct colonies from original plates (ompA, hybB, Aox1a, Aox1b) on 9/7/2010.

In an incubator tube add:

1. 5mL of LB media

2. add 5uL CARB

3. Add colony

4. incubate with shaking overnight at 37C

 

C) Miniprep of hybB (Gita, Christian, Scott)

Plasmid isolation protocol

1. Pellet 1.5 mL of cells by centrifuging 2-3 mins @ 3k RPM, and pour off remaining liquid
2. Resuspend pelleted bacterial cells in 250 µL Buffer P1 and transfer to a microcentrifuge tube.
3. Add 250 µL Buffer P2 and mix thoroughly by inverting the tube 4 - 6 times.
4. Add 350 µL Buffer N3 and mix immediately and  thoroughly by inverting the tube 4 – 6 times.
5. Centrifuge for 10 mins at 13,000 rpm (~17,900 xg) in a table-top microcentrifuge.
6. Apply the supernatants from step 5 to the spin column by pipetting.
7. Centrifuge for 30 – 60 secs. Discard the flow-through.
8. Wash spin column by adding 0.75 mL Buffer PE and centrifuging for 30 – 60 secs.
9. Discard the flow-through, and centrifuge for an additional 2 min to remove residual wash buffer.
10. Place the column in a clean 1.5 mL microcentrifuge tube. To elute DNA, add 30 µL autoclaved water to the center of each spin column, let stand for 1 min, and  centrifuge for 1 min. (NOTE: 50uL were added to elute)

 

Results of miniprep

HybB (two microcentrifuge tubes) is in the -20 in 218 (in a yellow container labeled Gaucher).

        Concentration

        hybB 1: 159.1 ng/uL

        hybB 2: 224.5 ng/uL

Concentration obtained using nanospectrometer in Gaucher lab (226).

 

Procedure:

1. Clean the sensor (center of the device where the arm meets the base) with a chemwipe.

2. Blank nanospec by pipetting 1uL of water (both water and pipet are next to the device) on to the sensor. Blow out the pipet so that the drop is hanging on the tip and touch it to the sensor rather than trying to drop the sample onto the sensor. Close the lever arm onto the sensor.

3. Click calibrate on the computer, wait for it to display 0ng/uL.

4. Clean sensor with Chemwipe.

5. Pipet 1uL of sample onto sensor, close lever arm.

6. Click measure and wait for sample reading.

7. Repeat 4-6 for each sample, clean sensor when done.

 

For next time-

1. Minipreps and cryostocks of the ompa, aoxa/b, pSB1A3
2. Start PCR steps for hyBb (getting it ready for the 5 constructs(

 

9/10/2010 (Scott, Debika, Christina, Margo, Gita)

Results and Observations:

        A) Triple smear: triple smear worked fine on the new plates with distinct colonies. Plates are in 4C fridge.

        B) Starter colonies from 9/9/10: Don’t appear very dense - interestingly the pSB1A3 starter colony didn’t turn red like it did last time.

 

Experimental Procedure:

        A) Miniprep of Aox1a, Aox1b, ompA, pSB1A3 from starter colonies from 9/9/10.

 

Miniprep

 

1. Remove inoculation tubes from inoculation (37C shaker).

2. Obtain P1 buffer from 4C refrigerator.

3. Take centrifuge tubes and add 1.5mL on inoculated cells.

4. Centrifuge at 3000 rpm (low) for 1-2 min.

5. Spin until white pellet of cells forms at the bottom and liquid is more clear.

6. Take off supernatant and discard.

7. Repeat steps 4-6.

8. Resuspend pelleted bacterial cells in 250սL P1 buffer.

9.  Add 250սL P2 buffer and invert 4-6 times (DO NOT VORTEX - doing so will shear DNA!)

10. Add 350սL buffer N3 and immediately invert 4-6 times.

11. Centrifuge for 10 min. at 13,000 rpm.

12. Take supernatant and add to spin columns.

13. Spin 30-60 sec. and discard flow through.

14. Wash column with 750սL buffer PE and centrifuge 1 min.

15. Discard flow through and centrifuge and additional minute.

16. Please column into a clean 1.5mL microcentrifuge tube.

17. Elute DNA by adding 30սL dH2O.

18. Let stand for 1 min., then centrifuge for 1 min.

Note for miniprep- I did 3 tubes for each plasmid, and I used 50 uL of water to elute DNA from one tube, then I used that 50 uL of already eluted DNA to elute the other two in series.

 

Making gel for PCR

1. Add 0.35g agarose to 35mL autoclaved water.

2. Add 3.5mL 1X TBE

3.  Heat in microwave until agarose dissolves. Allow to cool. Make sure there are NO VAPORS before adding EtBr. EtBr is an intercalator. Don’t vaporize it, especially near your face!

4. Add 38.5սL EtBr (edited from 45 uL, make 1000X)

5. Pour gel and allow to harden.

 

First Step for PCR (9/10/2010)

 

Labels on Tube:

           1= HybB F,R

        2= AOX1a F,R

        3= AOX1a F,R2

        4= AOX1b F,R

        5= AOX1b F,R2

        6= Ompa F, R

 

PCR Basic Recipe

        template DNA

        2 or more primers

        dNTPs

        buffer compatible with DNA polymerase being used

        DNA polymerase

 

Today, we didn’t have any RFP plasmid left, so we just did the following combinations:

        1 = HybB F, R primers with HybB template DNA

        2 = AOX1a F, R primers with AOX1a template DNA

        3 = AOX1a F, R2 primers with AOX1a template DNA

        4 = AOX1b F, R primers with AOX1b template DNA

        5 = AOX1b F, R2 primers with AOX1b template DNA

        6 = OmpA F, R primers with OmpA template DNA

 

The polymerase used in ALL tubes is the Phusion and the buffer used is the Phusion  MF Reaction Buffer . All components listed are stored in the iGEM freezer.

 

The steps for running PCR are as follows:

1. Obtain PCR tubes (very small, string of 8 tubes) with lids (also strung 8 together). Label. Obtain components. Buffer and template can be thawed at room temperature; enzymes and primers should be thawed on ice just long enough to obtain liquid & refrozen.

2. Primers used are 1:10 dilution. We prepped them in Eppe tubes, 20 uL into 180 uL for a total of 200 uL, stored in the freezer. For use in reactions, to avoid contaminating stock by repeatedly drawing from it.

3. Template concentration must be measured, and fall between 50 and 200 ng/uL. If prepared template does not fall in this range, adjust amount used in step 4e. below, and adjust water used in step 4a.

4. Add liquid components, keeping primers, dNTPs, template, and polymerase on ice (NOTE: components in steps c, d, e can be added in any order. Just be sure a & b go in first, and f goes in last.):

        a. 14 uL H2O

        b. 5 uL Phusion MF Reaction buffer

        c. 2.5 uL forward primer &

            2.5 uL reverse primer

        d. 0.5 uL dNTP - (thawed & kept on ice)

        e. 0.25 uL template DNA

        f. 0.25 uL polymerase enzyme, Phusion.

5. Volume should total 25 uL. Seal lids. Store all leftover components at -20 degree freezer. Run in PCR cycler.

 

Instructions for running PCR thermocycler:

1. The on/off switch is on the back right side. Turn it on. The machine has a graphic showing you how to open it. Basically, turn the top wheel counter-clockwise to open, push in.

2. Our cycle program is saved under RUN -> EAG1 -> MEGAN -> IGEM. Select. Tell it if you are using side A or B. Hit run.

3. When prepping, it will ask for approximate sample size. Saying 25 or 30 uL is fine. Leave SampleID and User blank. Select Heat lids to 105 C.

4. Load tubes, avoiding corner slots (if possible). Close lid, tighten clockwise until you feel minor resistance-- don’t crank too hard.

3. PCR cycler will hold at 12 degrees C after completion of cycles, until you pick it up (or someone else turns the machine off).

4. When you are done, if no one else is using the machine, turn it off.

 

Results (9.10.2010)

Miniprep-

 

Concentrations (ng/uL)

AOXa-350

AOXb-313

PSB1A3-107

ompA- 80

 

9/11/2010

Goals: Purify the PCR reactions and look at them on a gel

PCR purifcation of the PCR reactions from 9/10/2010

See protocols page for PCR purification

 

We purified both strips 1 and 2 from the PCR last night, they are labeled this way in the freezer:

 

1 HybB F, R

1 AOX1a F, R

1 AOX1a F, R2

1 AOX1b F, R

1 AOX1b F, R2

1 OmpA F, R

 

2 HybB F, R

2 AOX1a F, R

2 AOX1a F, R2

2 AOX1b F, R

2 AOX1b F, R2

2 OmpA F, R

 

As we were not certain that the PCR strip 1 had been successfully put together (may have doubled up on the buffer and neglected to add the enzyme), we decided to run the gel using just the samples from the PCR strip 2. We used only 5 uL of each, leaving the leftovers in the freezer for later reference or use.

 

Running an Agarose Gel to see reults of strip 2 PCR from 9/10/2010

1. After running PCR purification using QIAGEN kit (eluting at the end into water), prepared a gel with 12 wells. Prepared gel electrophoresis kit by placing fresh gel into well, and filling with 1X TBE up to the top of the gel.
2. Laid out a strip of clean parafilm, made discrete dots of 1 uL of dye for each sample (excluding ladder) being prepared.
3. Mixed 5 uL of DNA in water with 1 uL of loading dye for each sample, directly onto parafilm. Pulled up a total of 6 uL. Transferred to the gel in this order:

1. Lane 1: DNA Ladder (6 uL) (Pretty sure you mean 5 uL and not 56 uL. Edited by Debika @ 1:54 am on 9/12/10. Yes, thanks. Margo).
2. Lane 2: 2 HybB F, R
3. Lane 3: 2 AOX1a F, R
4. Lane 4: 2 AOX1a F, R2  
5. Lane 5: 2 AOX1b F, R
6. Lane 6: 2 AOX1b F, R2
7. Lane 7: 2 OmpA F, R
8. Lane 8: DNA Ladder (5 uL)

4. Plugged in gel electrophoresis kit, ran at 100 V for ~ 25 min.

        

 

Results 9/11/2010

1. We could not see bands for the PCR reactions, except for the 4th and 6th lanes (very faint bands, and not in the correct “spot”.

For next time-

1. We need to troubleshoot why we couldn’t see bands for the PCR reactions.

1. Possibilities:
2. Use smaller wells (we used 20 uL wells)
3. Use more DNA (we loaded 5 uL of PCR DNA)
4. Redo the PCR reactions

2. Scan the Gel Picture