Team:GeorgiaTech/WeekSeven

From 2010.igem.org

8/5/2010

Extracting pCOLD from cells

1. Re-suspended pellet in 250 սL PI buffer

2. Added 250 սL P2, inverted about 5 times

3. Added 350 սL N3, mix immediately, inverted 4-6 times

4. Centrifuged 10 min, 13k RPM

Enzyme digest

5. Poured supernatant into spin column

6. Centrifuged 30-60 seconds and discarded flow-through

7. Washed column with 750 սL PE and centrifuged 30-60 seconds

8. Discarded flowthrough, centrifuged 1 min

9. Eluted by placing column in new 1.5 mL tube, added 30 սL EB. Let stand 5 min.

10. Centrifuged 5 minutes, 5k RPM

8/6/2010

Prepared 1% agarose gels:

Added 180 mL 1x TBE and 1.8g agarose

Heated up until agarose was no longer visible

Let cool (approx. 10 min)

Added 180 սL Ethidium bromide (1000X)

PCR purifcation of the cell cultures from 8/5/2010

1. Added 5 volumes of Buffer PBI to 1 volume of the PCR sample and mix. It is not necessary to remove mineral oil or kerosene.

2. Checked that the color of the mixture was yellow (similar to Buffer PBI without the PCR sample).

3. Placed a spin column in a provided 2 mL collection tube.

4. To bind DNA, applied the sample to the column and centrifuge at 17,900g for 30 – 60 secs.

5. Discarded flow-through. Placed the column back in the same tube.

6. To wash, added 0.75 mL Buffer PE to the column and centrifuged for 30 – 60 secs.

7. Discarded flow-through and place the column back in the same tube. Centrifuged the column for an additional 1 min.

8. Placed the column in a clean 1.5 mL microcentrifuge tube.

9. To elute DNA, added 50 µL Buffer EB (10 mM Tris-Cl, pH 8.5) or water (pH 7.0 – 8.5) to the center of the membrane and centrifuged the column for 1 min. Alternatively, for increased DNA concentration, added 30  µL elution buffer to the center of the membrane, let the column sit for 1 min, and then centrifuged.

10. If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.

Gel Extraction Protocol

1. Excised DNA fragment from the agarose gel with a clean, sharp scalpel.

2. Weighed the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg or approximately 100 μL).

3. Incubated at 50ºC for 10 min (or until the gel slice had completely dissolved). To help dissolve gel, mixed by vortexing the tube every 2 – 3 min during the incubation.

4. After the gel slice has completely dissolved, checked that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose).

5. Added 1 gel volume of isopropanol to the sample and mixed.

6. Placed a QIAquick spin column in a provided 2 mL collection tube.

7. To bind DNA, applied the sample to the QIAquick column, and centrifuged for 1 min.

8. Discarded flow-through and placed QIAquick column back in the same collection tube.

9. Recommended: Added 0.5 mL of Buffer GQ to QIAquick column and centrifuged for 1 min.

10. To wash, added 0.75 mL of Buffer PE to QIAquick column and centrifuged for 1 min.

11. Discarded the flow-through and centrifuged the QIAquick column for an additional 1 min at 17,900 x g (13,000 rpm).

12. Placed QIAQuick column into a clean 1.5 mL microcentrifuge tube.

13. To elute DNA, added 50  μL of Buffer EB (1 mM Tris-Cl, pH 8.5) or water (pH 7.0 – 8.5) to the center of the QIAquick membrane and centrifuged the column for 1 min. Alternatively, for increased DNA concentration, added 30  μL elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuged for 1 min.

14. If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.

8/9/2010

Ligation

Purpose: Insert mRFP into pCold vector

In a microcentrifuge tube add:

1. vector DNA (pCold)

2. insert DNA (mRFP)

3. Ligase 10X buffer

4. T4 DNA Ligase

5. Nuclease-free water to a final volume of 10սL.

6. Incubate reaction at 4C overnight.

*(pCold: 5700 bp; mRFP: 700 bp)

Making LB media

To make 1L LB (Lysogeny broth) medium:

5g Yeast abstract

10g Tryptone

5g NaCl

To make solid plates, follow the above recipe and add 16g agar per 1L.

Prepared 2L of LB media, 1L liquid broth and 1L to make plates (don’t fill 1L bottles full - make 4 bottles 500 mL each).  

Autoclave, leaving lids slightly loosened.

Let cool in water bath at 55C.

Add antibiotic to LB media used for plates.

For the agar plates, pipetted 25mL into each plate

Incubating reactions

The bacteria were left at 4C overnight.

8/10/2010

Heat shock transformation of the plasmids into our bacteria

10 սL Nova Blue cells + 5 սL of ligation rxn

1. Left cells and ligation reaction products on ice.

2. Added plasmid (5սL) (ligation product) to cells. Mix gently by swirling pipet tip in mixutre (DO NOT ASPIRATE).

3. Left cells on ice for 10-30 min.

4. Applied heat shock of 45 seconds in 42C bath.

5. Put tubes on ice for 2 min.

6. Added 250 սL of LB (room temp.)

7. Incubated 1 hour at 75C (9/1 Edit: Seems improbably high- probably 37 C)

8. Plated 100սL, then remaining amount on separate LB/CARB plates.

9. Incubated overnight at 37C.

8/11/2010

Inoculation

1. Add 5mL LB and 5սL CARB into test tubes (16 total)

2. Pick 2 well-defined colonies from each plate (labelled sequentially).

3. With an inoculation loop, get 1 colony from transformation plate and place into test tube.

4. Repeat for each colony (16 total).

5. Inoculate overnight at 37C.

8/12/2010

Miniprep

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

2. Obtain P1 buffer from 4C refrigerator.

3. Take centrifuge tubes and add 1.5mL of 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.

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 an 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.

Double digest

Into an Eppendorf tube add:

1. ~ 25սL DNA

2. 10սL autoclaved H2O.

3. 4սL 10X buffer.

4. 0.5սL Nde1 enzyme.

5. 0.5սL Xho1 enzyme.

8/13/2010

Making gel for PCR

1. Add 0.5g agar to 50mL autoclaved water.

2. Add 5mL 1X TBE

3.  Allow to cool.

4. Add 45սL EtBr

5. Pour gel and allow to harden.

Running PCR

1. Fill gel to top with 1X TBE.

2.  Take 3սL loading dye and make a spot on parafilm.

3.  Add 10սL DNA to dye on parafilm.  Pipette up and down to mix (NO BUBBLES).

4.  Load 5սL of ladder at each end.

5.  Add DNA + dye to wells (total 13սL).

6.  Put lid on. Run at 100V, until about 1/2 way down the plate.

7.  Transport in tubberware to image.

8.  Lanes that appeared to “run” well after imaging were inoculated on 8/16/2010.

8/16/2010

Inoculation of cell cultures 5, 8 and 9

1. In a Eppendorf tube add 3սL carbinicillin (CARB) to 3 mL LB media.

2. Inoculate at 37C overnight.

8/17/2010

Cell culture tubes 5, 8, and 9 from 8/16/2010 were removed from inoculation and used for two purposes:

Freezing a portion of inoculated cells for future use

300 սL of glycerol were added to 200 սL of each culture and tapped gently until the culture had mixed with the glycerol. These were then marked and stored in the freezer in the upstairs lab.

Addition of IPTG to induce expression of pCOLD

To test the effects of IPTG on mRFP (and thus pCOLD) expression, we prepared 4 concentrations of IPTG for each of the 3 tubes of bacteria (5, 8, and 9), resulting in 12 tubes.

Each contained:

930սL LB media

50սL culture

10սL CARB

10սL IPTG

where the 10սL of IPTG were calculated serial dilutions insured to give an overall IPTG concentration of 0.1 M (10^-3), 1 mM (10^-5), 10 սM (10^-7), and 100 nM (10^-9) for each tube number 5, 8, and 9. The resulting 12 samples were put in the incubator at 37C overnight.

If everything goes according to plan, it should be possible to see some RFP expression tomorrow.

8/18/2010

Expression of mRFP should have been detectable by red coloring in the samples after incubation.  Nothing was seen, and as such the samples were discarded. WE PUT THEM IN THE WRONG CELLS! Novablue cells are inapropriate for this type of transformation... redo the transformation with BL21 cells.

8/19/2010

Transformation into BL21 cells

Adding IPTG to plates:

1.  Spread 50սL IPTG on 2 LB/carb plates (50սL each plate).

2. Wait 1 hour to allow IPTG to soak into media.

Preparing cells:

3. Chill 2 empty 1.5սL Eppendorf tubes, 1:4 ligation reaction product, and 20սL BL21 cells on ice.

4. Aliquot 10սL of BL21 cells into Eppendorf tubes (10սL each tube).

5. In each tube add 1սL 1:4 ligation reaction mixture.

6. Allow to sit on ice for 10-30 min.

7.  Place in 42C bath for 30-45 sec.

8. Immediately place back on ice for 2 min.

9. Add 250սL room temp. LB media to reaction tube.

10.  Places tubes in 37C incubator/shaker for 1 hr.

11. Spread 125սL of incubated cells onto labelled LB/CARB plates according to below:

12. Incubate overnight (16 hrs) at 37C.

8/20/2010

Cold shock:

1. Place places in appropriate temperature incubators (4C or 15C), and wait to see expression of RFP.

Results: One red colony observed on IPTG plate at 4C at 9:20 am.

8/23/2010

Cold shock results:

One red colony on IPTG plate at 4C (first observed at 9:20 am on 8/20/2010) and one red colony observed on IPTG at 15C (8/23/2010).

8/23/2010

Purpose: try to maximize expression of mRFP.

Making a “starter” culture:

1. From 4C NON-IPTG plate, take a white colony and place in approx. 3mL of LB/CARB media.

2. Incubate with shaking overnight at 37C.

8/24/2010

Purpose: from starter culture monitor OD to obtain OD600 of 0.4-0.5.

1. From starter culture take 50mL liquid culture and add 50uL CARB in a large Erlenmeyer flask.

2. To this, add 1mL from starter culture and begin monitoring OD (for each aliquot take 1mL for measuring OD); maintain flask at 37C incubation with shaking. Keep in mind that e-coli doubling time is 20 minutes.

3. Put on incubator/shaker at 11:20 am, with initial total volume of 51mL, and OD600 = 0.0.

4. Put incubator at 15C. During the cooling period, the flask was kept at 6C to ensure that the colony quit growing. Final OD was 0.57 before putting in the incubator (without shaking) for 30 minutes. (Time in: 1:30 pm). Total volume of the flask at this point was 47mL.

5. At 2:00 pm, remove flask from incubator and IPTG (100mM) to a final concentration between 0.1 and 1mM (choose 0.25 mM).  For a final concentration of 0.25mM IPTG in a 47mL solution, add 117.5uL of IPTG (100mM stock).

6.  Continue culture with shaking at 15C for 24 hours.

8/25/2010

Results from 8/24/2010: expression unsuccessful.

Start with fresh colonies from the plates from 8/19/2010.  Since we know the mRFP made it successfully into the pCold vector on the two colonies that turned red, try to make starter cultures from those.

Making a “starter” culture:

1. From 4C and 15C IPTG plate, take a red colony and place in approx. 3mL of LB/CARB media. (3mL LB + 3uL CARB).

2. Incubate the two tubes with shaking overnight at 37C.

Plan for 8/26/2010: Make cultures as we did on 8/24/2010 again, monitoring the concentration of cells.  Then run a miniprep to ensure that we have the mRFP inserted into the pCold vector.

8/26/2010

Successful growth of starter colonies. Repeat process from 8/24/2010 until cell concentration is OD600 = 0.4 to 0.5.

1. Prepare 2 flasks (1 for each starter colony) acccording to directions from 8/24/2010.

2. Repeat procedure from 8/24/2010. (final concentration of cells in each flask was approx. OD600 = 0.4). Amount of (100mM) IPTG added: 125uL to each flask.

3. Incubate with shaking overnight at 15C.

8/27/2010

WE GOT RED FLASKS!!!!!!!!!!!!

Now, we’re going to do a Miniprep and run an experiment analogous to what we did on 8/19/2010.

Miniprep of 2 cultures: one inoculated from a fluorescent colony on the 4º C plate; one inoculated from a fluorescent colony on the 15º C.  

1. Protocol from 8/12/2010
2. Concentrations:

Transformation of BL21 by pCOLD+mRFP (from 4C  and 15ºC cultures)

1. Plasmid, cells should be on ice initially

1. + 1uL plasmid into 20 uL of competent cells (BL21)
2. Sit on ice, 10 mins
3. place in 42º water bath, 30 seconds
4. place in ice, 2 minutes
5. +250 uL LB broth
6. Plate: spread 100 uL onto plate, use spatula to spread around until resistance is felt.

1. I plated an IPTG plate and a non-ITPG plate
2. The IPTG plate had 25 uL of 0.1 M IPTG

7. Plates were left (upside down) on bench at room temperature (since the starter culture is not needed until Sunday, and these cells were transformed today, Friday)

For Next Time:

Sunday: Start 5 mL culture with one fluorescent colony of pCOLD+mRFP

Monday: Protein Expression under various conditions (i.e. cold, iptg, cold+iptg) in 5x50mL flasks.

8/29/2010

Results from the plates of 8.27.2010 transformation (transformed BL21 with pCOLD+mRFP):

non-IPTG plate- growth, many colonies, some are slightly red

IPTG plate- growth, many colonies, many are red, there are smaller red colones in the center along with larger normal ones; the last observation. Ryan suggested that it may indicate iptg stressed the cells in the middle (since IPTG could be more concentrated in middle).

What I did:

MAKING A STARTER Culture: I picked a slightly red colony from the non-iptg plate and innoculated 5 ml of LB+Carboamp with it.

Also, I put the iptg plate into the 15º C incubator and left it overnight

8/30/2010          Margo, Scott, Debika, Christian, and Christina

Observations: the non-iptg had more red colonies than it previously did on 8/29/2010

Induction experiment: inducing pCOLD+ mRFP under 4 different conditions (in BL21)

We added 1ml of starter solution to 50 ml of LB media and 50 ul of Carb. Put it in the 37C incubator shaker for an hour and a half. Checked OD levels after 1.5hrs for one flask. Since it was at .3, we checked again in 10 minutes.

After 10 minutes, the ODs values were as follows:

We wanted OD values to be @ ~0.4 (we got this number from the pCold manual). Added IPTG to one flask and put it in the 37C incubator w/ shaking along with another flask that had no IPTG.

Took the two other flasks and put it in the 15 degrees incubator shaker. After half an hour, we added IPTG to one of the flasks at 15 degrees, and left all 4 flasks in the respective incubators overnight.  

DMSO Stock (General protocol, applied here to pCOLD+mRFP containing cells from 8/30/2010)
Add to a cyrovial:

900 uL starter culture (from 8/30/2010 which was already fluorescent)

100 uL DMSO

1. Vortex 5 seconds
2. Put in -80c freezer (Gaucher lab)

Transformation of BL21 by pCOLD+mRFP (from 4º C and 15ºC cultures)

2. Plasmid, cells should be on ice initially

1. 1 hour before transformation, spread 25 uL of 0.1 M IPTG onto one plate

2. + 1uL (1:1, ligation product) plasmid into 20 uL of competent cells (BL21) (edit:9.1.2010 the plasmid and cells should be on ice 15 mins before you start 2-8)

3. Sit on ice, 10 mins

4. place in 42ºC water bath, 30 seconds

5. place in ice, 2 minutes

6. +250 uL LB broth (edit 9/1/2010:  and wait 30 minutes @ 37C- tape down in incubator)

7. Plate: spread 100 uL onto plate, use spatula to spread around until resistance is felt.

1. I plated an IPTG plate and a non-ITPG plate
2. The IPTG plate had 25 uL of 0.1 M IPTG

8. Plates were left (upside down) in 37ºC incubator overnight, to be examined on Tues 31 Aug.

        Note: Plate in incubator in room 218.

8/31/2010

Results from the four flasks incubated overnight:

+IPTG 37 C: Deep red cultures (color seen within an hour)

no IPTG 37 C: No color

+IPTG 15 C: Light red cultures (color seen the next morning)

no IPTG 15 C: No color

Contents of flasks bleached and discarded.

pCold in solution doesn’t require cold shock for expression, just IPTG. However, as seen from 8/29, pCold without IPTG colonies turned red when plated. Ryan noted that cells respond differently when in bulk vs. colonies, though these differences can’t really be generalized.

Results from plates from the transformation conducted on 8/30/2010:

No colonies were observed on the plates left in the incubator, cause: lack of incubation (essential for recovery after LB added) during transformation. During all transformations, the incubation step is crucial. Plates discarded.

Plan:

1. Make more media.

2. Make more plates.

Making LB media and plates

To make 1L LB (Lysogeny broth) medium:

5g Yeast abstract

10g Tryptone

5g NaCl

Fill up to 1L with dH2O.

To make solid plates, follow the above recipe and add 16g agar per 1L right before autoclaving.

Prepared 2L of LB media, 1L to be used as liquid broth and 1L to make plates.

Let cool at room temp.

For plate preparation:

Add 1:1000 antibiotic (CARB - plates marked with blue line) after autoclaving and cooling.

-1mL antibiotic per 1L media

Pipette 25mL into each plate.

Plan for 9/1/10:

1. Retransform from 15C colonies (not forgetting incubation!) from a miniprep.

2. Grow up cell cultures of pCOLD+mRFP run the following 4 conditions (as with the bulk solutions observed this morning):

Further, another colony will be plated without IPTG and left at room temp. This might allow us to determine if there are repeatable differences between plate and bulk bacteria behavior. The red color from warm, NON-IPTG plates leads us to think that we might see red without IPTG, and the extra room-temp control is determine if the response is temp-dependent.

9/1/2010

11 am: Prepared IPTG plate for transformation (25 uL IPTG) (Scott)

Margo, Scott, Mitesh, Gita, and Debika

12 pm: Performed another transformation following protocol from 08/30/10. We transformed pcold+mrfp (pcold+mrfp plasmid, the 15c one from 8.27.2010). This time, we put both the non-iptg plate and iptg plate into a 15c incubator.

9/2/2010

Results: no cells grew on either plate from 9/1/2010.

Thew plates away on 9/7/2010.

3 pm: Gita and Christian

Objective: Determine if pCold is activated at intermediate concentrations of IPTG

Performed serial dilution of IPTG to make 1 mM, 10uM, and 100nM, 1mL stocks. Grew bacteria from DMSO stock starter-culture from 8/30 in test tubes and incubated in 37C and 15C shakers.

9.3.2010

To do:

-reconstitute aox, transform using NovaBlue

-also, transform Ompa, hybB (check cells to transform into)

Experimental things:

Plated some BL21 onto a plate that was made by iGEM, to test if something is wrong with the plates

Plate labeled: 9.3.2010 Igem BL21

Threw plates away on 9/7/2010.

Tuesday 9/7/2010

Where are the results from the experiment on 9/2/2010?

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

1. Left cells and ligation reaction products on ice.

2. Added plasmid (5սL) to cells. Mix gently by swirling pipette tip in mixture (DO NOT ASPIRATE).

3. Left cells on ice for 10-30 min

4. Applied heat shock of 45 seconds in 42C bath.

5. Put tubes on ice for 2 min.

6. Added 250 սL of LB (room temp.)

7. Incubated 1 hour at 37 C

8. Plated 100սL, then remaining amount on separate LB/CARB plates.

9. Incubated overnight at 37C.

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

1. Added 5 volumes of Buffer PBI to 1 volume of the PCR sample and mix (in a clean 1.5 mL eppendorf)

2. Checked that the color of the mixture was yellow (similar to Buffer PBI without the PCR sample).

3.. To bind DNA, transferred the sample to the column and centrifuge at 17,900g for 30 – 60 secs.

4. Discarded flow-through. Placed the column back in the same tube.

5. To wash, added 0.75 mL Buffer PE to the column and centrifuged for 30 – 60 secs.

6. Discarded flow-through and place the column back in the same tube. Centrifuged the column for an additional 1 min.

7. Placed the column in a clean 1.5 mL microcentrifuge tube.

8. To elute DNA, 30  µL autoclaved milliQ water to the center of the membrane, let the column sit for 1 min, and then centrifuged.

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

9/13/2010

Christina, Christian, Scott, Debika, Margo

Started from new aliquots of primers, 1:10 dilution of primer stock in MilliQ H2O. Changes are bolded.

Reactions: HybB F+R, OmpA F+R, AOX1A F&R, and AOX1B F&R.

New PCR protocol:

26.5 uL H2O

10 uL TaQ 5X Reaction buffer

5 uL forward primer

5 uL reverse primer

1 uL dNTP 10 mM - (thawed & kept on ice)

2 uL template DNA

0.5 uL polymerase enzyme, TaQ

Total Volume= 50 uL

NOTE: We multiplied the entire protocol by 2 to get 50  uL total volume for this attempt

We also decided to load just the plasmids onto gel TODAY, to check that our template stocks are fine and were not the issue with the PCR failure this past weekend.

We are reducing number of experiments at once (e.g. not all setups at once, just a few) - HybB, OmpA, AOX1A F&R, and AOX1B F&R.

We are preparing two strips for PCR using this recipe and setup. We are then running them side-by-side in the PCR machine, one strip using the old cycle program, and one strip with two key modifications (suggested by Megan). We increased number of PCR cycles from 29 to 34 cycles, and reduced annealing temperature to 52 degrees. (If we go to low with the annealing temperature, we will be able to tell because we will see lots of bands on PCR.)

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.

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

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

1. Added 5 volumes of Buffer PBI to 1 volume of the PCR sample and mix (in a clean 1.5 mL eppendorf)

2. Checked that the color of the mixture was yellow (similar to Buffer PBI without the PCR sample).

3.. To bind DNA, transferred the sample to the column and centrifuge at 17,900g for 30 – 60 secs.

4. Discarded flow-through. Placed the column back in the same tube.

5. To wash, added 0.75 mL Buffer PE to the column and centrifuged for 30 – 60 secs.

6. Discarded flow-through and place the column back in the same tube. Centrifuged the column for an additional 1 min.

7. Placed the column in a clean 1.5 mL microcentrifuge tube.

8. To elute DNA, 30  µL autoclaved milliQ water to the center of the membrane, let the column sit for 1 min, and then centrifuged.

note: unless we want to keep the DNA for future use (unless we NEED pure DNA), the purification step is not necessary.  We could have just run the DNA without the purification step... EtBr is an intercalator that will only bind the DNA anyway.

PCR Results via Gel Electrophoresis

Ran at 100 V for ~ 4 minutes, until dye was visible ~ 3/4 of the way across the gel. (This seems like it was too long!)

DNA was visible in all four lanes; two replicates of each lane (labeled A and H), with the A samples having been run with the “old” PCR cycle program, and the H samples having been run with the “new” PCR cycle program (as described by today’s changes).

1. HybB with Forward and Reverse primers

2. AOX1a with Forward and Reverse primers

3. AOX1b with Forward and Reverse primers

4. OmpA with Forward and Reverse primers

The A lanes and its DNA ladder were  more clearly visible than the H lanes. The visible results are:

1. Faint bands in A & H at ~ 6,000 bp

2. In A& H, very strong bands at 700 bp, strong bands at 3,000 bp, somewhat weak but  still obvious band at 2,000 bp.

3. In A& H, very strong bands at 700 bp, strong bands at 3,000 bp, somewhat weak but  still obvious band at 2,000 bp.

4. DNA may have “run off” the gel! In the H lane, distinct but faint band visible past the visible bands of the DNA ladder-- not sure how this should be interpreted.

(Pics from 9/11 and 9/13 were taped to the lab bench for group reference -- maybe get Megan and/or Richard to help us interpret.)

Starter cultures for cryostocks

Made starter cultures (3uL CARB + 3mL LB + cells) from triple smear plates (9/10/2010), and put in the incubator for 24 hours..  Tomorrow (on 9/14/2010), make cryostocks from these starter cultures. Labelled according to insert.

9/14/2010

Results from 9/13/2010:  The previous gel had fairly good AOX bands but after consulting with Richard we decided that the other samples weren’t represented in the gel. Further, we should have seen primer bands near the end, so in future gels it’s important not to let samples run off.

Miniprep

In order to prepare for another gel, a miniprep of the following samples from the starter cultures made on 9/13/2010 were run:

1. PSB1A3

2. AOX1a

3. ompA

4. hybB

5. AOX1b

The contents were labeled and stored in the -20 freezer for further use in the gel.

Crystocks

Cryostocks were made from starter cultures grown on 9/13/2010.  Two distinct colonies were taken from each plate - so there are duplicates of each.  Stored in the -80C labelled:

9-14 clg HybB (2)

9-14 clg ompA (2)

9-14 clg AOX1a (2)

9-14 clg  AOX1b (2)

9-14 clg psb1A3 (2) [note: 1 of these starter cultures turned red, the other did not.]

9.15.2010

Goals: Perform PCR on the new minipreps from 9/14/2010 (using new 1:10 aliquots of primers from 9/13/2010)

PCR Protocol

26.5 uL H2O

10 uL TaQ 5X Reaction buffer

5 uL forward primer

5 uL reverse primer

1 uL dNTP 10 mM - (thawed & kept on ice)

2 uL template DNA

0.5 uL polymerase enzyme, TaQ

Total Volume= 50 uL

Notes: Wear gloves while doing the reaction. Keep all reagents on ice, including the PCR reactions. Add in the order of the protocol- get out the enzyme and place on ice right before you are about to use.

Reactions Done in PCR:

1. HyBb F,R
2. OmpA F,R
3. Aox1a F,R
4. Aox1a F,R2
5. Aox1b F,R
6. Aox1b F,R2

Notes: We are using a master mix of Water, TAQ Buffer, DNTPS, and TAQ. Add this to all the tubes (everything on ice), then add all the DNA reagents.

Results from nanospec:

Sample                Concentration (ng/uL)

hybB                155.2

ompA                70.2

Aox1a                241.3

Aox1b                253.2

psb1A3                53.6

Making gel for PCR

1. Add 0.35g agarose to 36 mL 1 x TBEautoclaved water.

2.  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!

3. Add 35 սL EtBr (edited from 45 uL, make 1000X)

4. Pour gel and allow to harden.

9/16/2010

Goals:

1. Interpret gel results of the PCR from 9/15/2010
2. Perform PCR (from 9/15/2010) again, this time with:

1. PHUSION Polymerase/Buffer
2. RFP

What we did:

(Christina, Rob)

1. Performed PCR with Phusion
2. Reactions:

1. HyBb F,R
2. OmpA F,R
3. Aox1a F,R
4. Aox1a F,R2
5. Aox1b F,R
6. Aox1b F,R2
7. RFP F,R
8. RFP F2,R

7. Performed miniprep of pSB1A3 from cell culture grown on 9/15/2010 (non-red)

PCR Protocol

26.5 uL H2O

10 uL PHUSION 5X Reaction buffer

5 uL forward primer

5 uL reverse primer

1 uL dNTP 10 mM - (thawed & kept on ice)

2 uL template DNA

0.5 uL polymerase enzyme, PHUSION

Total Volume= 50 uL

Miniprep of pSB1A3

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

2. Obtain P1 buffer from 4C refrigerator.

3. Take centrifuge tubes and add 1.5mL of 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.

Cryostock of hybB cells and pSB1A3 (from cultures on 9/15/2010)

900 uL of cells

100 uL DMSO

Total 1 mL

Mix, place in -80c Freezer

For next time

1. Confirm all the predicted sizes match up with the observed sizes on the gel of the PCR from 9/15/2010
2. If all are confirmed (Christina has confirmed, Christian and Scott confirmed hybb, ompa), then proceed with PCR purifications of 40 uL of each reaction, then RE digests, then ligations to construct each construct

SEPTEMBER 17, 2010

Goals:

Since the PCR was run on 9/16 using Phusion (high fidelity), we need to:
A) Run gel (2 uL each)

B) PCR purify (leftovers from the 50 uL stock= 48 uL)

C) Nanospec

D) Digest with restriction enzymes (runs overnight)

Protocols

Make a gel for running PCR products from 9/16/2010 to make sure the PCR was successful.

Making gel for PCR (1% agarose gels):

Added 180 mL 1x TBE and 1.8g agarose

Heated up until agarose was no longer visible

Let cool (approx. 10 min) until there are NO MORE VAPORS

Added 180 սL Ethidium bromide (1000X)

Pour into gels and allow to harden (large wells hold about 35 mL).

Note: it is ok to make 6 gels at once according to the recipe above and store them. It is not necessary to “immediately” use gels as long as they are stored properly. From now on, when you make 1% gels, just fill all 6 wells at once.

Gel of the PCR from 9/16/2010 was run today and imaged:

Lane order: 100 bp ladder|aox1aFR|aox1aFR2|aox1bFR|apx1bFR2|HybB FR|ompa FR|RFP FR| RFP F2R|100 bp ladder

volumes:

loaded 4 ul of the aox reactions, 7 ul of ompa and hybB, and 5 ul of the RFP reactions

PCR Purification

1. Added 5 volumes of Buffer PBI to 1 volume of the PCR sample and mix (in a clean 1.5 mL eppendorf)

2. Checked that the color of the mixture was yellow (similar to Buffer PBI without the PCR sample).

3.. To bind DNA, transferred the sample to the column and centrifuge at 17,900g for 30 – 60 secs.

4. Discarded flow-through. Placed the column back in the same tube.

5. To wash, added 0.75 mL Buffer PE to the column and centrifuged for 30 – 60 secs.

6. Discarded flow-through and place the column back in the same tube. Centrifuged the column for an additional 1 min.

7. Placed the column in a clean 1.5 mL microcentrifuge tube.

8. To elute DNA, 30  µL autoclaved milliQ water to the center of the membrane, let the column sit for 1 min, and then centrifuged.

Nanospec results (PCR from 9/16/2010)

AOX1a-F,R = 99.3 ng/uL

AOX1a-F,R2 = 50.0 ng/uL

AOX1b-F,R = 48.5 ng/uL

AOX1b-F,R2 = 42.1 ng/uL

OmpA-F,R = 27.3 ng/uL

RFP-F2,R = 10.8 ng/uL

Hyb-F,R = 26.2 ng/uL

RFP-F,R = 8.8 ng/uL

Restriction Enzyme Digest General Process

- check buffers (NEB site)

-check if BSA required (NEB site)

- Start w/ HybB and RFP (start @ step A) / pBS1A3 (start @ step D)

Recipe for : 50 uL RXN

DNA (1-2 ug- based on nanospec results)

1 uL each enzyme

5 uL 10X buffer

1 uL BSA(if needed)

Today we are digesting just the HybB-F,R and RFP-F,R in order to do the ligation on this simple construct. The rest of the PCR-ed, purified, and nanoscpec’ed building blocks went into the freezer for later use.

RE Digest Recipe for HybB

40 uL HybB-F,R (based on the nanospec results above)

1 uL EcoRI

1 uL NotI

4.7 uL 10X buffer for EcoRI [Edit 9/17/2010. Only EcoRIbuffer)

1 uL 10x BSA (based on this specfic RE mix-- BSA is required)

Place into an eppe tube, pipet gently to mix. Incubate at 37 degrees in water bath overnight.

RE Digest Recipe for RFP-F,Rr

40 uL RFP-F,R (based on the nanospec results above) [Edit 9/17/2010, 110 ul not there, reduced to 40]

1 uL SpeI (edited changed from notI to speI )

1 uL NotI

5 uL 10X buffer for EcoRI [Edit, only EcoRI buffer]

1 uL10x BSA (based on this specfic RE mix-- BSA is required)

Place into an eppendorf tube, pipette gently to mix. Incubate at 37 degrees in water bath overnight.

Results

Gel of multiple PCR reactions

100 bp Ladder| Aox1a FR| Aox1a FR2| Aox1b FR| Aox1b FR2| HybB FR| Ompa FR| RFP FR| RFP F2R| 100 bp ladder|

For Future

1. We have a strategy for each construct, written on the board. We must digest each PCR product to build our building blocks, then construct each construct through a series of ligations, digest, PCRs etc (See 9.18.2010 for strategy)

9.18.2010

Goals

1. PCR purify the hybB F,R and RFP F,R (rxns from 9/16/2010)
2. Ligate hybB F,R and RFP F,R

Notes:

1. RFP F,R was accidently digested with NotI and not SpeI.
2. What to do? We will need to add 1 uL of SpeI and run overnight, so that the SpeI site is cleaved.
   Just in case we need this RFP, we PCR-purified it today and stored in freezer, clearly labeled PARTLY DIGESTED RFP. If we need to use this RFP, it will need to be digested by SpeI.
3. RFP band on the gel pic from 9/17/2010 was faint and nanospec showed low conc (8.8 ng /uL) versus the higher yields of the other rxns.

Ran PCR purification on HybB, stored in freezer. This HybB was nanospec’ed on Friday and had a concentration of  26.2 ng/uL. This has been digested by the right RE’s, EcoRI and NotI, and ready to be ligated with RFP when it has been appropriately digested.

PCR Purification

1. Added 5 volumes of Buffer PBI to 1 volume of the PCR sample and mix (in a clean 1.5 mL eppendorf)

2. Checked that the color of the mixture was yellow (similar to Buffer PBI without the PCR sample).

3.. To bind DNA, transferred the sample to the column and centrifuge at 17,900g for 30 – 60 secs.

4. Discarded flow-through. Placed the column back in the same tube.

5. To wash, added 0.75 mL Buffer PE to the column and centrifuged for 30 – 60 secs.

6. Discarded flow-through and place the column back in the same tube. Centrifuged the column for an additional 1 min.

7. Placed the column in a clean 1.5 mL microcentrifuge tube.

8. To elute DNA, 50  µL autoclaved milliQ water to the center of the membrane, let the column sit for 1 min, and then centrifuged.

BASIC PLAN FOR BUILDING PLASMID CONSTRUCTS

from the primered building blocks, which are stored in our freezer

A) RUN GEL - 2 uL each - to check size - this step completed for all building blocks

B) PCR PURIFY leftovers - this step completed for all building blocks

C) NANOSPEC - record concentrations - this step completed for all building blocks

D) DIGEST with restriction enzymes, overnight

E) PCR PURIFY digestion products

F) LIGATION of gene building blocks - we are starting with HybB & RFP

G) PCR ligation products, HybB-RFP

H) DIGEST products, HybB-RFP

I) RUN GEL on small amount of digestion products, HybB-RFP

J) PCR PURIFY the rest of the digestion products, HybB-RFP

K) DIGEST the vector - we are using pBS1A3

L) PCR PURIFY the vector

M) LIGATION of gene with vector, HybB-RFP and pBS1A3

N) TRANSFORMATION of plasmid into E. coli, run overnight

Note- Do a nanospec after PCR purifications

NOTES:

1. Steps in BOLD are going to be repeated with each plasmid construct. Steps A,B, and C have already been done for each building block and won’t be repeated unless there is a specific issue with a particular building block.
2. Steps K and L do not necessarily need to wait until step J is completed to be run. They are listed this way so there is no confusion about what is being digested or PCR purified at each step.

Plan for this week:

Weekend: PCR purify hybB, run gel for pSB1A3

1. Mon: Redo PCR RFP; digest RFP (O/N)
2. Tues: ligate hyB+RFP (O/N), Digest pSB1A3 (O/N)
3. Wed: PCR ; digest (1hr); PCR purify (digests of hyb+rfp and pSB1a3) at same time we run gel to check results; ligate O/N
4. Thurs: Tranformation of cells by hyb+RFP+psb1a3
5. Fri: check plates

9/20/2010

Goals:

1. Redo PCR of mRFP F,R; mRFP F2,R
2. Digest mRFP F,R; mRFP F2, R O/N (not possible until we get enzymes)
3. RUN GEL - 2 uL each - to check size - this step completed for all building blocks
4. PCR PURIFY leftovers - this step completed for all building blocks
5. NANOSPEC - record concentrations - this step completed for all building blocks
6. Transform novablue with mRFP that ryan gave us (labeled mRFP H) to create our own crytostock

Notes:

1. Ryan gave us a fresh stock of mRFP plasmid. We only get this one! It is precious like gold or diamonds!
2. Since the original mRFP has a Nde I site in the MCS, we are worried other products may run at the same size. To avoid this worry, we will use gel extraction to extract only our band of interest.

mRFP

1. in the plasmid PET15B
2. Use Novablue to create a cryostock. (transform, grow on plate, pick colony, grow in liquid media, take cryostock)

Protocols

9am

PCR Protocol for mRFP F,R; mRFP F2, R

26.5 uL H2O

10 uL PHUSION 5X Reaction buffer

5 uL forward primer

5 uL reverse primer

1 uL dNTP 10 mM - (thawed & kept on ice)

2 uL template DNA

0.5 uL polymerase enzyme, PHUSION

Total Volume= 50 uL

Ran PCR results (pre purification) on gel)

12pm

Heat shock transformation of the plasmids into our bacteria

10 սL Nova Blue cells + 5 սL of MRFP plasmid (from stock that Ryan gave us this morning).

1. Left cells and ligation reaction products on ice.

2. Added plasmid (5սL) to cells. Mix gently by swirling pipette tip in mixture (DO NOT ASPIRATE).

3. Left cells on ice for 30 min.

4. Applied heat shock of 45 seconds in 42C bath.

5. Put tubes on ice for 2 min.

6. Added 250 սL of LB (room temp.)

7. Incubated 1 hour at 37 C

8. Plated 100սL and left plate in the 37 degrees incubator  

9. Incubated overnight at 37C.

Results

1. Gel picture of mRFP FR, F2R (Insert gel pic!)

1. The bands of both PCRs (pre digest) were between approximately around  700-800

Gel of mRFP FR and mRFP F2R

100bp ladder|mRFP FR|mRFP F2R|Control

PCR Purification-mRFP

1. Added 5 volumes of Buffer PBI to 1 volume of the PCR sample and mix (in a clean 1.5 mL eppendorf)

2. Checked that the color of the mixture was yellow (similar to Buffer PBI without the PCR sample).

3.. To bind DNA, transferred the sample to the column and centrifuge at 17,900g for 30 – 60 secs.

4. Discarded flow-through. Placed the column back in the same tube.

5. To wash, added 0.75 mL Buffer PE to the column and centrifuged for 30 – 60 secs.

6. Discarded flow-through and place the column back in the same tube. Centrifuged the column for an additional 1 min.

7. Placed the column in a clean 1.5 mL microcentrifuge tube.

8. To elute DNA, 30  µL autoclaved milliQ water to the center of the membrane, let the column sit for 1 min, and then centrifuged.

Nanospec Results

mRFP, F: 137.2ng/uL

mRFP, F2: 132.3 ng/uL

For Future

1. Digest first thing Tuesday!
2. Richard suggested getting two squirt bottles- 1 for water, 1 for bleach (to kill cells)
3. Check if transformation of Nova Blue with mRFP worked!

9/21/2010

Results from 9/20/2010

        Transformation of mRFP in NB cells successful (both plates).

Starter Cultures

Make starter cultures from both mRFP:NB plates, allow to grow overnight.

3mL LB + 3uL 1000X CARB

put in 37C incubator with shaking overnight

Received shipment of SpeI (200 uL) and XmaI (50 uL) from Promega, put in -20C freezer (Gaucher Lab).

For 9/22/2010:

1. Begin digests 9am, do 3 hours insteadf of overnight, and ligation in evening
2. create cryostocks from starter cultures grown on 9/21/2010.
3. Couldn’t start RE digest of RFP today- could not find RE’s. Will ask Megan Wednesday morning.

9/22/2010- (Mitesh, Scott, Gita)

Goals

1. Digest of RFP F,R
2. PCR purify the digest
3. Ligate hyBB FR, to RFP F,R

Notes: made a stock of 1 ug/ul BSA for easier RE digests

I am following the suggested RE protocol listed on the Promega literature that came with the RE’s

RE Digest Recipe for RFP-F,Rr

4.5 uL H20

2uL 10X Promega Buffer D

10 uL RFP-F,R ( from 9.20.2010, 137 ug/ul)

2 uL BSA (0ug/uL, to a final conc of .1mg/ml)

0.75 uL SpeI (edited changed from notI to speI )

0.75 uL NotI

Total=20 ul total

Run 3 hours in the heating blockin our lab (37c, put water in the heating blocks)

Start time is 10:35 am

End time should be 1:35 pm

Purification of RFP digest

1. Added 5 volumes of Buffer PBI to 1 volume of the PCR sample and mix (in a clean 1.5 mL eppendorf)

2. Checked that the color of the mixture was yellow (similar to Buffer PBI without the PCR sample).

3.. To bind DNA, transferred the sample to the column and centrifuge at 17,900g for 30 – 60 secs.

4. Discarded flow-through. Placed the column back in the same tube.

5. To wash, added 0.75 mL Buffer PE to the column and centrifuged for 30 – 60 secs.

6. Discarded flow-through and place the column back in the same tube. Centrifuged the column for an additional 1 min.

7. Placed the column in a clean 1.5 mL microcentrifuge tube.

8. To elute DNA, 30  µL autoclaved milliQ water to the center of the membrane, let the column sit for 1 min, and then centrifuged.

Nanospec

RFP Digest (purified)- 41 ng/uL

Ligation of RFP-FR to HybB-FR

using hybB from 9/18/2010 and RFP from 9/21/2010

Calculating equivalents:

RFP- [41 ng/uL]/678 bp= 0.06 eq/uL

hyBb-[26 ng/uL]/393 bp = 0.06 eq/uL

for linear ligations, use a 1:1 ratio of products

Ligation:

2 uL of RFP FR (SpeI, NotI digested; purified)

2 uL of HybB FR (EcoRI, NotI digested; purified)

1 uL 10x Ligase Buffer

4.5 uL H20

0.5 uL T4 Ligase

Total=10 uL

Leave RT for 1 hour

Start time: 3:20 pm

End time: 4:20 pm

PCR of hybB+Mrfp construct

3 uL of ligation reaction

25.5 uL H2O

10 uL PHUSION 5X Reaction buffer

5 uL forward primer

5 uL reverse primer

1 uL dNTP 10 mM - (thawed & kept on ice)

0.5 uL polymerase enzyme, PHUSION

Total Volume= 50 uL

Start: 5pm

End: Thursday

For Future

1. Take out pcr of mrfp+hybB
2. Run 2 uL on gel, along with just the RFP-FR and hybB-FR (1hr)
3. Digest the construct, along with the vector psb1a3 at the same time (3 hrs)
4. Ligate the construct to psb1a3 (1 hr)
5. Transform into e coli (1.5 hrs)

9.23.2010 (Scott, Rob, Gita)

Goals

1. Run PCR of RFP+hyBb on gel
2. Digest the construct, along with vecotr psb1a3
3. ligate the construct to psb1a3
4. transform into e. coli

Notes:

Predicted size of hyb+MRFP~1000bp

Protocols

Running PCR product on gel

1. The pcr was left overnight at 12 c, and I am running 4 uL on a gel
2. 4  uL sample + 1 uL running dye

Notes:

1. I could not see any stained DNA, even the ladder. This means the EtBr degraded in the gels. Solution, suggested by Richard, is to keep a bottle of 1% Agarose/TBE solution at RT. This solution represents the first step of making a gel. Then whenever we need a gel, we start from the pre-made solution, add Etbr, and procede as normal. Start with fresh gels.
2. PCR products store in yellow box
3. Building blocks in blue rack

Making gel for PCR

1.  Heat 30 mL of 1% agarose solution  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!

2. Add 35 սL EtBr (edited from 45 uL, make 1000X)

3. Pour gel and allow to harden.

Results

Gel from 9.23.2010.

100 bp ladder| RFP-FR +HybB PCR|

Predicted Size- 1070 bp

Due to multiple bands, we decided to gel extract the construct RFP+hyBB. To do this, we borrowed a larger Gel try and apparatus from the Hammer Lab (they had a box of things they were not using). The larger gel is approximately 80 ml and is larger in area. The new gel will help make cutting the band easier.

Making gel for PCR (Hammer Lab Apparatus)

1.  Heat 90 mL of 1% agarose solution  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!

2. Add 90 սL EtBr (edited from 45 uL, make 1000X)

3. Pour gel and allow to harden.

(I did 100 mL, but found the actual volume to be around 80 to 90 mL)

Start: 4:10 pm

End: 5:10 pm

Gel Extraction Protocol

1. Excised DNA fragment from the agarose gel with a clean, sharp scalpel.

2. Weighed the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg or approximately 100 μL).

3. Incubated at 50ºC for 10 min (or until the gel slice had completely dissolved). To help dissolve gel, mixed by vortexing the tube every 2 – 3 min during the incubation.

4. After the gel slice has completely dissolved, checked that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose).

5. Added 1 gel volume of isopropanol to the sample and mixed.

6. Placed a QIAquick spin column in a provided 2 mL collection tube.

7. To bind DNA, applied the sample to the QIAquick column, and centrifuged for 1 min.

8. Discarded flow-through and placed QIAquick column back in the same collection tube.

9. Recommended: Added 0.5 mL of Buffer GQ to QIAquick column and centrifuged for 1 min.

10. To wash, added 0.75 mL of Buffer PE to QIAquick column and centrifuged for 1 min.

11. Discarded the flow-through and centrifuged the QIAquick column for an additional 1 min at 17,900 x g (13,000 rpm).

12. Placeed QIAQuick column into a clean 1.5 mL microcentrifuge tube.

13. To elute DNA, added 30  μL water (pH 7.0 – 8.5), let the column stand for 1 min, and then centrifuged for 1 min.

Gel of RFP-FR + HybB FR PCR product (Before Gel Extraction)

30 uL of PCR product|9.5 uL of 100 bp ladder

Band to be excised is the 1100 bp band (2nd bright band from bottom)

Gel of RFP-FR + HybB FR PCR product (After Gel Extraction)

30 uL of PCR product|9.5 uL of 100 bp ladder

For Future-

Friday-

1. nanospec the gel extracted hyb+RFP construct (in blue box)
2. Run 2 uL on gel, see if 1100 bp is confirmed
3. if so, continue with strategy.

9/24/2010Goals

4. nanospec the gel extracted hyb+RFP construct (in blue box)
5. Run 2 uL on gel, see if 1100 bp is confirmed
6. if so, digest the construct and vector psb1a3 (3 hrs); ligate (1hr), transform (1.5 hours)

Protocols

1 % Agarose Gel

1. I found that the small gel trays need only 25-30 mL of solution (less than 30)
2. I heated 30 mL of 1 % Agarose solution for approximately 45 seconds. I then added 30 uL of EtBr (use blue gloves) and poured into the prepared gel tray (tray inside of assembly, with combs). Cool for 1 hr. The gel will be done when there is a faint blue hue visible when looking at it.
3. Gel Start: 9:10 am
4. End 10:10 am

Nanospec of Gel Extract from RFP+HybB PCR

19.2 ng/uL

(inset gel pic from etbr lab)

Gel of Excised 1100 bp band of  RFP-FR + HybB FR PCR product

4 uL of PCR product|9.5 uL of 100 bp ladder

Band to be excised is the 1100 bp band (2nd bright band from bottom)

RE Digest Recipe for HybB +RFP PCR (Mitesh did this today)

3.5 uL H20

3uL 10X Promega Buffer E or Multicore

20uL HybB+RFP (excised 1100 bp badn from PCR) ( from 9.23.2010, 19 ng/ul)

3 uL BSA (1ug/uL, to a final conc of .1mg/ml)

0.75 uL SpeI

0.75 uL EcoRI

Total=30 ul total

Run 3 hours in the heating blockin our lab (37c, put water in the heating blocks)

RE Digest Recipe for pSB1A3 (Debika did this today)

3.5 uL H20 (edit 9/24/2010- should be 8.5 uL)

5uL 10X Promega Buffer E or Multicore

10 uL pSB1A3 (9.16.2010, 100 ng/uL)

5 uL BSA (1ug/uL, to a final conc of .1mg/ml)

0.75 uL SpeI

0.75 uL EcoRI

Total=50 ul total

Run 3 hours in the heating block in our lab (37c, put water in the heating blocks)

PCR Purfiy the two digests (RFP+Hyb construct, pSB1A3)

Note- We have had the best results when eluting in 30 uL of H20

Nanospec

pSB1A3 Digest (purified) = 6.2 ng/uL

RFP+HYBB Digest (purified)= 4.4ng/uL

Ligation of pSB1A3 to [RFP-FR+ HybB-FR] (from 9.23.2010)

Calculating equivalents:

pSB1A3- [ 6.2 ng/uL]/ 2100 bp=  3x10^-3 eq/uL

hyBb+RFP-[ 4.4 ng/uL]/1100 bp = 4x10^-3  eq/uL

1. For plasmid to linear ligations, use a 1:2 ratio of vector:products if the products are purified (1:4 if not)

1. E.g. 1 equivalent of vector to 2 equivalent of linear product

Ligation Protocol:

3 uL of RFP + HYB (~ 2 to 3 uL)

2 uL of  Plasmid pSB1A3 (~ 1/2x amount of linear product on eq/uL basis)

1 uL 10x Ligase Buffer

3.5 uL H20

0.5 uL T4 Ligase (enzyme-- keep in freezer-- add last of all)

Total=10 uL

Leave RT for 1 hour

Start: 5:23 pm

Transformation

Heat shock transformation of the plasmids into our bacteria

10 սL Nova Blue cells + 5 սL of Ligation Reaction

1. Left cells and ligation reaction products on ice.

2. Added plasmid (5սL) to cells. Mix gently by swirling pipette tip in mixture (DO NOT ASPIRATE).

3. Left cells on ice for 30 min.

4. Applied heat shock of 45 seconds in 42C bath.

5. Put tubes on ice for 2 min.

6. Added 250 սL of LB (room temp.)

7. Incubated 1 hour at 37 C

8. Plated 100սL (x2 plates) and left plates in the 37 degrees incubator  

9. Incubated overnight at 37C.

Notes for Future-

1. Aliquots of BL21 and Novablue are in our -70c freezer
2. Autoclave fresh pipette tips- there is confusion over which tips fit what pipettes- we need to calibrate to make sure our tips and pipettes are giving us the volumes we want

SEPTEMBER 25, 2010

Goals-

1. Begin digesting the building blocks necessary to create the rest of the constructs
2. Pick colonies from the pSB1A3+Hyb+RFP transformation and grow overnight

Nanospec results (From tubes dated 9/17/2010)

AOX1a-F,R = 99.3 ng/uL

AOX1a-F,R2 = 50.0 ng/uL

AOX1b-F,R = 48.5 ng/uL

AOX1b-F,R2 = 42.1 ng/uL

OmpA-F,R = 27.3 ng/uL

RFP-F2,R = 10.8 ng/uL

Hyb-F,R = 26.2 ng/uL

RFP-F,R = 8.8 ng/uL

RE Double Digest Recipe for RFP-F2,R

0 uL H20 (plenty in the RFP-F2,R tube, which is very dilute)

5 uL 10X Promega Buffer D

40 uL RFP-F2,R ( from 9.17.2010, 10.8 ng/ul)

5 uL BSA (1ug/uL, to a final conc of .1mg/ml)

0.75 uL SpeI

0.75 uL NdeI

Total=51.5 ul total

Start :4pm

End: 7 pm

RE Double Digest Recipe for OmpA F,R

1.5 uL H20

5 uL 10X Promega Multi-Core Buffer

37 uL OmpA-F,R ( from 9.17.2010, 27.3 ng/ul)

5 uL BSA (1ug/uL, to a final conc of .1mg/ml)

0.75 uL NotI

0.75 uL XmaI

Total=50 ul total

Went in at 2:55 pm

RE Double Digest Recipe for AOX1a F,R

12.5 uL H20

3 uL 10X Promega Buffer B

10 uL AOX1a-F,R ( from 9.17.2010, 99.3 ng/ul)

3 uL BSA (1ug/uL, to a final conc of .1mg/ml)

0.75 uL SpeI

0.75 uL XmaI

Total=30 ul total

Went in at 3:05 pm

RE Double Digest Recipe for AOX1a F,R2

2.5 uL H20

3 uL 10X Promega Multi Core Buffer

20 uL AOX1a-F,R2 ( from 9.17.2010, 50 ng/ul)

3 uL BSA (1ug/uL, to a final conc of .1mg/ml)

0.75 uL NdeI

0.75 uL XmaI

Total=30 ul total

Start 4 pm

RE Double Digest Recipe for AOX1b F,R

2.5 uL H20

3 uL 10X Promega Buffer B

20 uL AOX1b-F,R ( from 9.17.2010, 48.5 ng/ul)

3 uL BSA (1ug/uL, to a final conc of .1mg/ml)

0.75 uL SpeI

0.75 uL XmaI

Total=30 ul total

Went in at 3:10 pm

RE Double Digest Recipe for AOX1b F,R2

0 uL H20

3 uL 10X Promega Multi Core Buffer

22.5 uL AOX1b-F,R ( from 9.17.2010, 42.1 ng/ul)

3 uL BSA (1ug/uL, to a final conc of .1mg/ml)

0.75 uL NdeI

0.75 uL XmaI

Total=30 ul total

Start 4 pm

Run 3 hours in the heating blockin our lab (37c, put water in the heating blocks)

Start time is 3, 4pm (two sets of reactions, noted in the individual descriptions)

End time should be 6,7 pm

Preparing liquid culture of pSB1A3+hybB+RFP

1. 3 mL of LB + 3 uL of Carb into a culture tube. Picked 2 colonies from plates from 9.24.2010
2. O/N at 37c in our incubator-shaker

For Future-

1. PCR purify the digests
2. start first round of ligations
3. PCR the ligation products

9/27/2010

Goals

1. PCR purify digests of building blocks from 9/25/2010
2. Start first round of ligations for each construct
3. Check ligations on gel
4. Digest; troubleshoot ligations if necessary

Protocols

PCR Purification of Digests from 9/25/2010 (Ompa FR , AOX1a/b FR and FR2, RFP F2R)

Reactions I am purifying:

RFP-F2R (NdeI, SpeI)

OmpA FR (NotI, XmaI)

Aox1a-FR (Xma, SpeI)

Aox1a-FR2 (Xma, NdeI)

Aox1b-FR (Xma, SpeI)

Aox1b-FR2 (Xma, NdeI)

1. Added 5 volumes of Buffer PBI to 1 volume of the sample and mix (in a clean 1.5 mL eppendorf)

2. Checked that the color of the mixture was yellow (similar to Buffer PBI without the PCR sample).

3.. To bind DNA, transferred the sample to the column and centrifuge at 17,900g for 30 – 60 secs.

4. Discarded flow-through. Placed the column back in the same tube.

5. To wash, added 0.75 mL Buffer PE to the column and centrifuged for 30 – 60 secs.

6. Discarded flow-through and place the column back in the same tube. Centrifuged the column for an additional 1 min.

7. Placed the column in a clean 1.5 mL microcentrifuge tube.

8. To elute DNA, 30  µL autoclaved milliQ water to the center of the membrane, let the column sit for 1 min, and then centrifuged.

Nanospec of Purified digests

RFP-F2R (NdeI, SpeI)= 28.6 ng/uL

OmpA FR (NotI, XmaI)= 15 ng/uL

Aox1a-FR (Xma, SpeI)= 12 ng/uL

Aox1a-FR2 (Xma, NdeI)= 25 ng/uL

Aox1b-FR (Xma, SpeI)= 23 ng/uL

Aox1b-FR2 (Xma, NdeI)= 21.3 ng/uL

Ligations

hyb= [26.2 ng/ul ]/ 393 bp = 0.067 eq/uL

ompa = [15 ng/uL]/81 bp = 0.185 eq/uL

Ligation of HyBb+ Ompa

5.7 uL H20

2 uL of HybB

0.8  uL of Ompa

1 uL 10x Ligase Buffer

0.5 uL T4 Ligase (enzyme-- keep in freezer-- add last of all)

Total 10 uL

RT for 1 hr. - Start time: 11:25 am

Aliquot T4 Buffer:

Make aliquots (5 uL) of the T4 Ligase Buffer so as to not continuously repeat freeze-thaw cycles.

USE FROM ALIQUOTS FROM NOW ON - NOT THE GREEN-CAPPED EPPENDORF.

Miniprep of pSB1A3+hybB+ompA

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

2. Obtain P1 buffer from 4C refrigerator.

3. Take centrifuge tubes and add 1.5mL of 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.

Making a 1 % gel

25*4=100 mL

We found that we had extra left over.

Start at 1:13 pm

End: 2:13 pm

PCR of Hyb+OmpA ligation

3 uL of product of ligation reaction

25.5 uL H2O

10 uL PHUSION 5X Reaction buffer

5 uL HybB-F forward primer

5 uL OmpA-R reverse primer

1 uL dNTP 10 mM - (thawed & kept on ice)

0.5 uL polymerase enzyme, PHUSION

Total Volume= 50 uL

Start: 12:57 pm

End: 3:00 pm (approx)

Plan:

Once PCR is finished, run 4 uL of the reaction on the gel. Check for a band running at a size of 393+81=474 bp, so approximately mid 400-500 bp. If band is observed, prepare for the digest (check for the RE sites based on what primers were used).

Gel Picture

insert gel pic from computer

band around 500 bp.

Nanospec PCR

Christina

326 ng/uL

9/28/2010

Goals

1. The gel picture of the hybB+ompa is in the folder.

1. The band is bright at 500 bp, and our predictions confirm the results.
2. PCR purify the PCR (in the yellow box)
3. Nanospec

Notes

1. Christina+Rob PCR purified the hyb+ompa PCR.
2. Richard suggested we can PCR Aox+RFP, then pcr that to the hyb+ompa. This wil take care of two constructs. The other two require Aox a/b to be attached to the hybb+ompa construct
3. For the constructs that have Aox+RFP, we can do a ligation of Aox to RFP; then, PCR that part and ligate it to the Hyb+ompa.

1. For the AOX-RFP constructs:

1. Ligate Aox1a-FR2 to RFP-F2R
2. LigateAox1b-FR2 to RFP-F2R
3. PCR each ligation reaction
4. PCR purify
5. Check results on gel

1. Gel extract if PCR results in multiple bands

6. Digest the Aox-RFP construct
7. PCR purify
8. Ligate the Aox-RFP digests to Hyb-Ompa digest
9. PCR the entire constructs

2. For the HybB-Ompa-Aox constructs:

1. Ligate the HybB-Ompa to Aox1a-FR or Aox1b-FR
2. PCR the constructs
3. PCR Purify
4. Check results on gel

1. if multiple bands, gel extract

5. Ligate Hyb-Ompa-Aox to pSB1A3
6. Transform into cells or PCR entire vector

Constructs

9/29/2010

Goals

1. Ligate Aox1a-FR2 to RFP-F2R (1hr)
2. LigateAox1b-FR2 to RFP-F2R (1hr at same time as step 1)
3. Digest HybB.Ompa (3 hours)

3. Ligate the HybB-Ompa to Aox1a-FR  
4. Ligate the HybB-Ompa to Aox1b-FR

4. PCR each ligation reaction (3 hr, run simultaneously)

1. Use Phusion Polymerase
2. Only 3 ul or so is needed for the PCR
3. PCR volume can be 30 or 50 uL (50 works fine and gets us lots of DNA)

5. PCR purify (45 mins max)
6. Check results on gel (40 mins max)

1. Gel extract if PCR results in multiple bands (1hr)

7. Digest the Aox-RFP construct (3 hours or overnight)
8.  

Protocols

Make sure all products are digested

1. hyb.ompa is not digested, so start that today

Ligation of AOX1a-FR2 to RFP-F2R (Scott)

using AOX1a-FR2 from 9/27/2010 and RFP-F2R from 9/27/2010

Calculating equivalents:

RFPF2R- [ 29 ng/uL]/678 bp= 0.0428 eq/uL

AOX1a-FR2-[ 25 ng/uL]/1035 bp = 0.0242 eq/uL

for linear ligations, use a 1:1 ratio of products

Ligation:

1.1 uL of RFP F2R

2 uL of AOX1a-FR2

1 uL 10x Ligase Buffer

5.4 uL H20

0.5 uL T4 Ligase

Total=10 uL

Leave RT for 1 hour

Started 10:42 am

Ligation of AOX1b-FR2 to RFP-F2R (Scott)

using AOX1a-FR2 from 9/27/2010 and RFP-F2R from 9/27/2010

Calculating equivalents:

RFPF2R- [ 29 ng/uL]/678 bp= 0.0428 eq/uL

AOX1b-FR2-[ 21.3 ng/uL]/1047 bp = 0.0203 eq/uL

for linear ligations, use a 1:1 ratio of products

Ligation:

1 uL of RFP F2R

2 uL of Aox1b-FR2

1 uL 10x Ligase Buffer

5.5 uL H20

0.5 uL T4 Ligase

Total=10 uL

Leave RT for 1 hour

Started 10:42 am

Digest of HybB.Ompa (Christina)

17 uL of Hyb.Ompa (58.2 ng/uL)

8.5 uL h20

3 uL 10x Buffer B (Promega)

0.75 uL EcoRI

0.75 uL XmaI

Total= 30 uL

37c heating block (with water) for 3 hours

Start: 10:00 pm

End: 1pm

Do the ligations once Hyb.Ompa is digested:

Ligation of AOX1a-FR to HybB.Ompa

using AOX1a-FR from 9/27/2010 and Hyb.Ompa from 9/29/2010

Calculating equivalents:

HybB.Ompa - [ ng/uL]/474 bp= eq/uL

AOX1a-FR-[ 12 ng/uL]/1035 bp =  0.0116 eq/uL

for linear ligations, use a 1:1 ratio of products

Ligation:

uL of Aox1a-FR

uL of Hyb.Ompa

1 uL 10x Ligase Buffer

uL H20

0.5 uL T4 Ligase

Total=10 uL

Leave RT for 1 hour

Ligation of AOX1b-FR to HybB.Ompa

using AOX1b-FR from 9/17/2010 and HybB.Ompa from 9/29/2010

Calculating equivalents:

HybB.Ompa- [ ng/uL]/474 bp= eq/uL

AOX1b-FR -[ 23 ng/uL]/1047bp = 0.022 eq/uL

for linear ligations, use a 1:1 ratio of products

Ligation:

uL of Aox1b-FR

uL of Hyb.Ompa

1 uL 10x Ligase Buffer

uL H20

0.5 uL T4 Ligase

Total=10 uL

Leave RT for 1 hour