Team:SUSTC-Shenzhen-B/week 3/8

August

Abstract

This week we tried to mutate the vector however we failed.

Week 3

8.12.2012-8.18.2012

8.12.2012

We did nothing.

8.13.2012

We started to learn how to do our experiment under the guide of a warmhearted laboratory technician.

Here is the whole process of our experiment.

a. Centrifuge the test tube which contains DNA sequence for about 5 mins. This is to prevent DNA from escaping when we open the tube.

b. Add water to dilute the DNA sequence. The concentration is 100μM.

c. Oscillation and centrifugation.

d. Take out 10μL of the liquid and dilute it to 100μL.

e. Make a template for PCR.

f. Use PCR.

g. Electrophoresis.

h. After half an hour, take photos to check whether PCR production is successful.

We do this to mutate the enzyme restriction sites.

8.14.2012

We realized yesterday’s PCR production failed after checking the photos we took and decided to repeat the experiment with another polymerase because we don’t know what went wrong. But this experiment failed again.

We tried to do PCR again with the third polymerase. But this time we added another primer as a control group to see whether there is something wrong with our primer. To be surprised, we saw the right stripe we wanted on the picture.

I have been anxious about our experiment for days and finally had a good night today.

8.15.2012

We did an enzyme digestion to examine whether the mutation is completed. Here are the steps:

1. Design a 10μL system.

2. Put it into a incubator which has a constant 37℃ temperature.

3. Do an electrophoresis.

From the picture, we can see there was another stripe. Compared to the previous PCR products, we concluded that the mutation was finished.

Then we use an enzyme named Dpn1 to cut the template into small pieces. Because the template is methylated and the PCR product is not, it’s safe to do so. The purpose of this step is to protect the template plasmid from duplication and transcription. Our next step is genetics transformation. The essence of this step is to put the plasmid into the body of the E.coli. So we put the petri dish into the 37℃ incubator and wait the E.coli’s reproduction.

8.16.2012

1. We used PCR to amplificate GFP and RFP.

2. We did an electrophoresis to see whether the PCR is successful.

3. Then we did a restriction enzyme digestion to cut the GFP & RFP.

4. After one hour, we did an electrophoresis to see whether the digestion is successful. However, today’s experiment is different from we did before. This gel is different because it can be recovered.

8.17.2012

We pick up the colony from E.Coli, then did a restriction enzyme digestion to see whether it is mutate.

To be disappointed, the mutation was failed. That is to say, we have to do it again. After all those failures, we realized that we have to face disappointment when doing experiment. We can’t be strong enough until we meet lots of difficulties. But the good thing is we’ve learned a lot of skills which will be very helpful for our future experiments.

8.18.2012

We came back to the school on Friday night.

We had a meeting on Saturday, in which we discussed what we did and what we’re going to do. It was very helpful. We felt more united after the meeting.

The freshmen all came to the school over this weekend. It’s our duty to help them. So we got very busy. But that was fun.