MAKING NEW NAD6- F, R AND PRESENTATION

April 04^th

Today we were going to make another PCR reaction, but we realized we didn’t have NAD6- F, R. But neither Mr. Adams nor I knew where the new bunch of NAD6 was located. Took us a while to find them so we looked for help to guide us through making some NAD6 from the concentrated bunch.

According to another researcher in the building we needed to make a mixture that we can use in an experiment with 9 part pure water and 1 part NAD6. If we put too little water the concentration wont bind the DNA, if the water is too much the solution will be diluted and we wont be able to work with it.

So to make a solution enough for the tubes, we needed 50 parts of NAD. So I made a mix of 45 part Water and 5 parts NAD6. So with this mixture next week we can make another PCR reaction put it under the same conditions in the Thermo cyclers and see how they work out.
Afterwards, Mr. Adams and I went over some ideas for my presentation, whether it be in a poster or during assembly. We went over some basic ideas that are crucial to mention in the presentation, the history of how the species were categorized before. We talked more about the morphology hypothesis and  examine the moths so that I know what their difference are physically.
Next week I am to bring a camera so that I can take some photos to include in my presentation. Its been a great semester at the internship, I have and am continuing to learn so much about science, research,lab work and my future plans to work in this field of science (lab/research). 

MORE MORPHOLOGY

March 29^th

Today we couldn’t make any PCR reaction. So we talked about the history of the species we are working with. Currently we are working with 3 groups, Xylis, Zale and Safia. Which were all placed in the ZALE group, and our work is to place them in their rightful taxas.

The species we work with in the lab however are all labeled ZALE until we get the sequence results and place them correctly. Last year Aileen Aisenberg made a "family tree" for the species and placed the ones she worked with in their right place. We are going to work a another group and do the same thing Aileen did last year, and by the end of the research we will hopefully be able to place all the species in their correct taxa. 

We didn’t do much today that I didn’t mention in other posts. In future posts I will hopefully have more details to fill up.

However, the morphology of the moths, seems very complicated, not easy to understand as they have so many small parts with complicated names.

SPRING BREAK

March 8th and 15th .
I didn't have internship in both these weeks because we were on Spring break.
Next week will continue making gel and designing PCR experiments.

AM BECOMING A RESEARCHER.

March 22^nd

Today Mr. Urban leaves us, so from now on its just Mr. Adams and me. I made gel to run the last PCR experiment, which was 5 microliter, so I added 1 microliter of dye. After running the gel we found that 3 of the new species (Safia) worked well with the conditions, but we couldn’t take a picture because the Electrophoresis didn’t work. After we checked that the species worked, I designed another experiment to run on Thursday.

The specimen that worked from last week was sent for sequencing so we simply have to wait for results so we can place the species in their right group of species in the family tree. The species that didn’t work are ZAL 2, 6 and 16 (Barcita phaegrapha). Mr. Adams suggested that we re-run the species that didn’t work and see which conditions will work better for them.

Now that I can prepare the PCR by myself I feel more confidant that ever being in a lab. I can even create experiments, decide amounts of ingredients needed for each and run them all by myself. After the results from sequencing we will be working more on the morphology part of the species than the molecular data set.

POSITIVE RESULTS FINALLY

March 01^st.

      Today I made a gel by myself and Ms. Urban watched as I was putting it together to make sure I was doing everything right. I followed the Instructions on measuring the agarose, buffer and microwaving time in intervals then carefully adding Ethidium Bromide. Afterward I cross-linked the little dish to put the PCR reaction in while the gel was cooling. I then prepared the gel rig and poured water in it before setting the gel in. Afterwards I spun my PCR reaction from last week and added 1 microliter of the blue dye before putting it in the gel underwater in the gel rig.

      The process that was once almost impossible for me to accomplish in an hour took only a few minutes to do. I set the PCR reaction into the gel and turn the gel rig on then waited for 5 minutes to let the EtBr bind and the electricity flow through the gel.

      After five minutes I took the gel to the UV table to take a picture and see if the condition we set from last week worked. And the results were marvelous. The conditions worked perfectly, for the second group; with decreased water and MgCl₂. As shown below the bands we very clear and there wasn’t much smug in them.

KEY:

White bands= Positive results

Red-Orange smug= Dyes

Dark bands= original gel wells

   

As any lab experiment to verify that the conditions work and will work for the species we set up another experiment with the same conditions. The PCR reaction was small and using the same species. I left the reaction in the thermo cycler with the 51 ⁰ C annealing temp. In two weeks I will make another gel and see if it worked.

PCR REACTION IN ONE HOUR

Feb 23^rd

      Last experiment didn’t work very well with the species we used and/or the annealing temperature so this time we used three new species; ZAL 70, 71, 72. Mr. Adams and I reduced the water by a factor of 1.7 so that the concentration of the reaction remains the same. Instead of the regular 17.5 Microliters for a 25 Microliter reaction we made 14. 8. With this change we also increased the amount of MgCl₂ from 1.5 to 2.5 Microliters. In addition we made another row of regular water amounts so that we have a better idea of which conditions work best for the species.

      With practice, this time I put the two PCR reactions in an hour instead of the regular two. I am much faster with the lab equipment and putting together the experiment. I also know my way around fixing small mistakes such as Pipette usage or controlling amounts of Primers, Q-taq, dNTP’s, Buffer and water to get good results.

      With the remainder of my time in the lab I learned to Morphological way of identifying the species I work with in their molecular level. Mr. Adams introduced me to the shapes of Moths under the microscope and their morphology as a whole. The best way to identify the Species and place them in the right taxa is in fact through their reproductive system the shapes of the male genitalia helps place it in the right taxa; same thing with females. 

MAKING GEL

Feb 16^th 2012

Today I made gel with Ms. Urban. On the station that we made the gel in was instructions with exact measurements to make the gel. 1g of Agarose, 500ml of water and 65 ml of buffer. Then them mixture is heated in a microwave to 1mnt 30secs. Afterwards 1 microliter of Ethedium Bromide (C₂₁H₂ON₃Br) is added so that it can bind the DNA together; the mixture is then poured into casting trays with combs when the PCR product will be put in.

After 5 minutes of cooling, the gel is put into gel rig for electrolysis. The gel rig is filled with buffer before putting the gel in, allowing the electricity to pass through. After another 5 minutes, the gel rig is turned off, and the gel with PCR reaction is taken for observation. We got 1 positive result for the experiment, on ZAL 74 with optimum temperature of 51⁰C. With the results 51⁰C should be the optimal temperature for our next experiment to get positive results. Also the positive result in this experiment was the one with increased amounts of MgCl₂, Primers and Taq. Next time though, we could also increase the primer a bit more because the band we got was a little faded. And the Thermo cycler program would be NAD51L for the new temperature, and we would add 5 cycles to make 39 cycles instead of 34.

With this lab experience, I have learned names of new machines and procedures that will surely be useful later. I measure the agarose in a special weigh scale; I put the PCR reaction into the gel in the gel rig. All these things are important for any lab work and because I plan to work on a lab this is a very valuable learning experience for me. 

NEW EXPERIMENT WITH CORRECTIONS

Feb 09^th 2012

I made another experiment today with corrections from last week’s mistakes. Instead of making 2 master mixes for 32 tubes; Ms. Urban suggested we make 4 big master mixes, 1 big tube for every 4 smaller tubes. After a little discussion, we also decided to make the standard MgCl₂ 2 microliters instead of the original 1.5. Other changes remain the same as last week.

We also set up the tubes in gradients as A1, B2, C3, D4, E4, F5, G6, and H7. This way when they are in the Thermo cycler group ZAL 76 of the standard and the altered group (D4 and E4), will kind copy each other. They will get the same temperature so we can really see the difference in the other groups.  The temperature of the Thermo cycler remained QIA42-57, meaning that the temperature with decrease from right to left (indicated with arrows on the right). The experiment will look like this in terms of temperature control and standard vs. altered group:

REGION

PRIMER

1

2

3

4

5

6

7

8

9

10

NAD 6

NAD 6 F-NAD 6 R

A

ZAL 73

ZAL 73

ZAL 73

C-

B

ZAL 74

ZAL 74

ZAL 74

C

C

ZAL 75

ZAL 75

ZAL 75

C

D

ZAL 76

ZAL 76

ZAL 76

C

NAD 6

NAD 6 F-NAD6 R

E

ZAL 76

ZAL 76

ZAL 76

C

F

ZAL 75

ZAL 75

ZAL 75

C

G

ZAL 74

ZAL 74

ZAL 74

C

H

ZAL 73

ZAL 73

ZAL 73

C

Next week, I am going to set up gel for my experiment.

SECOND EXPERIMENT AND MATHEMATIC CHANGES

Feb 02^nd 2012

Today Ms. Urban wasn’t in so I worked with Mr. Adams on another experiment. The previous one, my first experiment didn’t have good results; in fact, it didn’t work at all, no DNA was copied. So we’re to set up another one with some adjustments that Ms. Urban made to the “recipe”. This time we were to use 4 samples of DNA, the same ZAL group 13, 14, 15, and 16. The first experiment used ZAL 17, 74, 73, 76 and 75. We were also to use the same part of the DNA, NAD6. The Thermo cycler program however remained the same QIA42-57.

Before we began to make the master mix, Mr. Adams explained that they changed some of the recipe so that it matches the “original” experiment by some Brazilian researchers, he gave the published paper they wrote for me to get a better idea of what he was talking about.

The changes to the recipe was as follows:

PER 1 RXN	ORIGINAL AMMOUNT (microliters)	AMMOUNT IN STANDARD *4(microliters)	ALTERED (microliters) *4
Pure H2O	17.5	70	70
Buffer	2.5	10	10
MgCl2	1.5	6	(2.5)*4=10
dNTPs	2.0	8	8
Primers	.5	2	(.75)*4=3
Polymerase	.15	.6	(.2)*4=.8
TOTAL PER RXN		23		26.2
DNA		2		2

Unfortunately, Mr. Adams and I made a mistake and did not multiply the given amount by 4 to make the master mix and we ended up with very little mixture and could only distribute to 2 tubes. The total of the mix should’ve been about 386.4 microliters for the standard experiment and about 407.2 microliters for the altered mixture. Because there were 32 tubes, 16 for each group of experiment if we needed to multiply each amount to fit 16 tubes.

Even though we didn’t really make the mixture, I gained a bit more experience on how to use pipettes and it was great to make mistakes because it helps me learn and I can be more careful next time. We noted the mistake and next week we are to work on making the experiment correctly.