Operation Plasmid Rescue!

From each of the three plates of growth two colonies (one white and one red) were grown over night in YPAD at 30*.  The cells were then spun down and the broth was removed from the cells. Buffer P1 (250 micro liters) and glass beads were mixed with the cells and shook for ten minutes.  Buffer P2 (250 micoL) was added to the tube and mixed.  Then after 4 minutes buffer N3 (350 micoL) was added and mixed.  The tube was then centrifuge at 13,000 rpm for 10 minutes.  The top liquid was then removed and place in a spin column and spun for a minute (at 13,000 rpm).  The DNA was then washed with PB (500 microL) and centrifuged for a minute.  Buffer PE (750 mL)was added and centrifuged for a minute.  The column was then spun dry for a minute.  Buffer EB(30 microL) was added and sat for five minutes.  The DNA was then spun down into a clean tube.

Before we could send our plasmids in to be coded we had to amplify the DNA.  Taq master mix (9 microL), plasmid DNA from above (1 microL), Primer mix Seq (1 microL), and water (7 microL) were mixed and PCRed with the following program:

95* for 2 minutes

(95* for 30 seconds, 54* for 30 seconds, 72* for 1 minute) 35 times

72* for 10 minutes

4* forever.

 

The ender PCR was then ran on a gel check and then purified.

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Transformations (done on 3-10)

Only Constructs 10, 15, 20 and 25 yielded promising results from the two piece PCR so those are the ones we transformed onto yeast cells.

The procedure for transformation was as follows:

1) Spin yeast for five minutes that has been growing overnight in growth media at 30° Celsius.

2) Take the pellet that forms after spinning the yeast and place it in a 1.5 mL tube, put TE in the tube, and spin it for 30 seconds at 13k RPM.

3) pipet off the supernatant and Re-suspend the resulting pellet in 300 µL of TE with 50 µL of salmon sperm DNA that has been boiled for 5 minutes and then placed on ice. This solution will be called “cells” for the next table

4) Label 1.5 ml tubes 2p-10, 2p-15, 2p-20, 2p-25, neg cntrl, and add the corresponding things to each:

2p-10 2p-15 2p-20 2p-25 Neg cntrl
-40 µL of construct 10 -40 µL of construct 15 -40 µL of construct 20 -40 µL of construct 25 -40 µL of TE buffer
-8 µL of PER 154 -8 µL of PER 154 -8 µL of PER 154 -8 µL of PER 154 -8 µL of PER 154
-50 µL from cells tube -50 µL from cells tube -50 µL from cells tube -50 µL from cells tube -50 µL from cells tube
-150 µL of 1M Lithium Acetate* -150 µL of 1M Lithium Acetate* -150 µL of 1M Lithium Acetate* -150 µL of 1M Lithium Acetate* -150 µL of 1M Lithium Acetate*
-1 mL 50% PEG** -1 mL 50% PEG** -1 mL 50% PEG** -1 mL 50% PEG** -1 mL 50% PEG**

*Vortex the tubes quickly after adding the Lithium Acetate

**Vortex after adding the 50% PEG and mix by inversion a couple of times.

5) Incubate the tubes at 30° Celsius with rotation for 30 minutes.

6) Incubate for 15 minutes at 42° Celsius in a water bath.

7) Spin down the tubes at 13k rpm for 1 minute, remove the supernatant and re-suspend the pellet in 200 µL sterile dH2O

8) Plate each sample at 1x (190µL of resuspended cells) and 1/20x (10 µL of resuspended cells/The rest of the cells in the tube) onto SC-leu plates.

9) Allow the yeast to grow at 30° Celcius.

 

 

Below are pictures of some of the 1x (hi) plates, the low plates did not show many results.

Transformation Construct 25 hi Transformation Construct 15 hi Transformation Construct 20 hi

2 Piece PCR.

For the first 2 piece PCR we ran we used the result of the initial 10 PCR Constructs we made (5 N-terminal and 5 C-terminal) and then combined the corresponding construct numbers so that we would ultimately end up with 5 constructs. To make each of them we did the following:

Construct  10 Construct 15 Construct 20 Construct 25 Construct 30
.5 µl of EDR 301 .5 µl of EDR 301 .5 µl of EDR 301 .5 µl of EDR 301 .5 µl of EDR 301
.5 µl of EDR 262 .5 µl of EDR 262 .5 µl of EDR 262 .5 µl of EDR 262 .5 µl of EDR 262
2.0 µl of Construct 10 N terminal 2.0 µl of Construct 15 N terminal 2.0 µl of Construct 20 N terminal 2.0 µl of Construct 25 N terminal 2.0 µl of Construct 30 N terminal
2.0 µl of Construct 10 C terminal 2.0 µl of Construct 15 C terminal 2.0 µl of Construct 20 C terminal 2.0 µl of Construct 25 C terminal 2.0 µl of Construct 30 C terminal
25 µl of GoTAQ (Master Mix) 25 µl of GoTAQ (Master Mix) 25 µl of GoTAQ (Master Mix) 25 µl of GoTAQ (Master Mix) 25 µl of GoTAQ (Master Mix)
20 µl h2O 20 µl h2O 20 µl h2O 20 µl h2O 20 µl h2O

 

Construct 10 and 15 PCR

From my new N and C construct (post on 02/24/2013)  I first had to cut the DNA out of the gel and purify it using a gel purification kit. Then I made a 1/10 dilution of my N and C terminals.  For my two different constructs I added the following to each tube: 

10N

15N

10C

10N

1 microliter 1/10C

1 microliter 1/10N

1 microliter 1/10C

1 microliter 1/10C

1 microliter EDR 302

1 microliter EDR 302

1 microliter 1866 primer

1 microliter 1867 primer

1 microliter 1677 primer

1 microliter 1679 primer

1 microliter EDR 262

1 microliter EDR 262

9 microliter Master mix

9 microliter Master Mix

9 microliter Master Mix

9 microliter Master Mix

8 microliter H2O

8 microliter H2O

8 microliter H2O

8 microliter H2O

 

I then ran the PCR for the following time:

95 ˚C for 30 seconds

     One time

95 ˚C for 30 seconds

54 ˚C for 30 seconds

72 ˚C for 1 minute

     Twenty-five times

72 ˚C for 10 minutes

     One Time

10 ˚C forever

 

First I did a test 1% agrose gel but then decided that because the bands were a little messy I would run all of the products into the gel and cut out the top band of each construct (Figure 1).  Lastly I purified the DNA using a gel purification kit.

 Image

Figure 1: from left to right: 500BP ladder, 10N construct, 15N construct, 10C construct, 15C construct in a 1% agarose gel for 45 minutes at 100V.   

PCR of 5 N/C mutant constructs

From the previous PCR, the dominant bands were cut out of the agarose gel and purified in order to be used in five different constructs (10, 15, 20, 25, and 30) of the N and C terminals. The five C terminal constructs were made of 1.0 μL dil. 1×10 C terminal reactant, 9.0 μL GoTAQ master mix, 8.0 μL sterile water, and 1.0 μL EDR 262 antisense primer into each C terminal construct; 1.0 μL of sense primers 1866, 1867, 1868, 1869, and 1870 were added to C terminal constructs respectively. The five N terminal constructs were made of 1.0 μL dil. 1×10 N terminal reactant, 9.0 μL goTAQ master mix, 8.0 μL sterile water, and 1.0 μL EDR 302 sense primer into each N terminal construct; 1.0 μL of antisense primers 1677, 1679, 1681, 1683, and 1685 were added to N terminal constructs respectively.
The reaction mixture underwent PCR as follows:
95 ˚C for 30 seconds
     One time
95 ˚C for 30 seconds
54 ˚C for 30 seconds
72 ˚C for 1 minute
     Twenty-five times
72 ˚C for 10 minutes
     One Time
10 ˚C forever
Figure 1: N terminal PCR products ran at 100 volts through 1% agarose gel for 30 minutes with an anomaly of 2 bands

Figure 1: N terminal PCR products ran at 100 volts through 1% agarose gel for 30 minutes with an anomaly of 2 bands

Figure 2: C terminal PCR products ran at 100 volts through 1% agarose gel for 30 minutes with an anomaly of 2 bands
Figure 2: C terminal PCR products ran at 100 volts through 1% agarose gel for 30 minutes with an anomaly of 2 bands

 

N and C Terminal 2.0

We decided that for accuracy reasons that we want to repeat the experiment from the beginning.  We also decided that all three of us are going to work on our own to make our work more accurate by being able to compare our work with each other.  So we made the primer mixes with 1 microliter of the one of the two terminal primers and diluted it with 4 microliters of water.  Then in each of PCR mixes we added 8 microliters of the Master Mix, 0.5 microliters of the 1/20x per556, 1 microliter of the primer mix, and 6 microliters of dH2O.  The PCR program was the same as our previous time ( see post from 2/14/2013).  For this gel we put in two ladders, a 500 base pair ladder and a 100 base pair ladder.  The gel was also ran for an hour instead of half hour like the previous PCR, so we could get a more accurate estimate of the size of the section of the DNA.  In the picture below the first well had the 500 bp ladder, the next well had the N terminal, then the C terminal and the last we had the 100 bp ladder.

Image

PCR of N and C Terminal

Before we can start working with yeast we have to mutate DNA to cross over into the yeast. The first step was to replicate the N and C terminal of the sup35 portion of the DNA.  We made primer mix for the N terminal with 2 microliters EDR257, 2 microliters EDR260 and 8 microliters water.  For the C terminal we mixed 2 microliters EDR654, 2 microliters EDR304, and 8 microliters water.  Then we made the mix to be PCRed with 18 microliters HiFi super mix, 1 microliter 1/20x pER556, 2 microliters primer mix, and 15 microliters sterol water.  The PCR time is the following:

95*C for 2 minutes

(95*C for 30 seconds, 54*C for 30 seconds, 72*C for 1.5 minutes) 30 times

72*C for 10 minutes

10* forever

After the PCR we ran the DNA through an agarose gel to see if there was DNA.  In the picture below is the DNA in the gel.  On the left is the N terminal and on the right is C terminal.  Our PCR appears to be successful.

PCR