Team:SUSTC-Shenzhen-B/week 4/8

August

Abstract

We tried to mutate the vector again and we failed again.

Week 4

8.19.2012-8.25.2012

8.19.2012

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.

8.20.2012

1. A senior student did a PCR for the template plasmid over the weekend. Then she did an electrophoresis. We can see from the photo that the duplication was successful. She also made a reaction system to do the restriction enzyme digestion. We used enzyme Afl II. To make all of us satisfied, the mutation was successful. She also transformed the plasmid into the body of a competent cell, and let it grow for one night.

2. The next step is to amplify the GFP and RFP which is not difficult. Here are our reaction systems.

Taq 0.25ul Taq 0.25ul 1 94℃

Buffer 2.5ul Buffer 2.5ul 2 cycle 94℃

Dntp 2ul Dntp 2ul 3 cycle 55℃

G-SXA-R 1ul R-NPS-R 1ul 4 cycle 72℃

G-SXA-F 1ul R-NPS-F 1ul 5 72℃

817 1ul 817 1ul

ddH20 17.25ul ddH20 17.25ul

3.After we amplified the GFP and RFP, we began to digest GFP with SpeⅠand AflⅡthese enzymes; RFP with NotⅠand SpeⅠthesis enzymes. Let me write our reaction system clear:

H buffer 2ul M buffer 2ul

BSA 1ul Spe 1 1ul

Not 1 1ul Afl 2 1ul

Spe 1 1ul GFP 4ul

RFP 4ul dd H2O 12ul

dd H20 11ul

4. After the digestion, we did an electrophoresis, then purified the gel. Today we got the GFP and RFP with the right restriction enzyme cutting sites.

5. We picked up the colonies from E.coli.

8.21.2012

We did a restriction enzyme digestion with the colonies we picked up yesterday to see whether the mutation plasmid has been duplicated. 6 out of all 8 samples are successful.

8.22.2012

At first, we digested the plasmid with Not I and Afl II. Then we did an electrophoresis and purified the gel to get the production – plasmid cut by enzymes. After that, we connected the GFP, RFP, and plasmid together under 16℃ environment.

8.23.2012

We transformed the new plasmid into the body of E.coli and waited the bacteria to grow. Since we don’t have such type of experience, a teacher showed us the process before we started.

8.24.2012

Since it might be difficult to connect three fragments together at the same time, we decided to try connecting GFP and the plasmid at first, then connecting the product with RFP.

To start this new scheme, we have to digest the plasmid again but with different restriction enzymes. We used Spe I and Afl II to cut the plasmid.

Because we run out of GFP and RFP, we did the PCR again to get more.

The E.coli which contains our new plasmid – all 3 fragments connected – grows well. We did a bacterial colony PCR, using the RFP’s upstream primer and GFP’s downstream primer, to check whether the connection is successful. If the amplification of RFP and GFP is successful, we can come to a conclusion that we finally build the right vector we want!

8.25.2012

Today is Saturday. We did a PCR again to prove that the connection is Ok.

We’ve already digested the plasmid last week. Our next step is to digest GFP and RFP at the same time.

We’re going to check whether it is feasible to connect 3 fragments at the same time.