User:Lu Wang/Notebook/Team Allergy/2010/06/24

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Today's objective is to obtain purified DNA of the arabadopsis allergen panel. The last attempted to PCR the allergen genes was unsuccessful, possibly due to the Phusion Polymerase warming up too much. This time, the reaction will be repeated and kept on ice throughout the entire procedure.

Once we obtained purified genes, we will proceed to digestion with Xba1 and Pst1, ligation into V0120 vectors, and transformation of the vector into E. coli.

Our procedures for today were...

  1. PCR of arabadopsis allergen panel
  2. Nanodrop of PCR products
  3. Electronic Gel of PCR products
  4. Purification of PCR products
  5. Nanodrop of purified genes

Our E. Gel did not run properly, so we decided to run a traditional gel of the purified PCR products. But because we wanted to minimize DNA loss, we ran another PCR reaction (PCR #2) of the purified DNA from the first PCR reaction (PCR #1). This way, we would only lose a small amount of purified DNA but have enough DNA to see the results clearly on a gel.

  1. PCR #2 of purified allergen genes
  2. Gel Electrophoresis of PCR #2 products


PCR of arabadopsis allergen panel

Like last time, we followed the [Silver: PCR] protocol. This reaction was successful for all of the allergen genes. This reaction was kept on ice throughout the entire procedure, unlike last time's, and we feel that the temperature difference was key in the success of this reaction.

Nanodrop of PCR products

Our concentrations after PCR in (ng/μL) were:

Gene PCR #1 Products Purified DNA
LTP 1 sense 220.9 20.4
LTP 1 antisense 62.6 12.2
Bet v1.1 sense 216.2 15.4
Bet v1.1 antisense 228.1 11.5
Bet v1.2 sense 205.5 9.2
Bet v1.2 antisense 68.5 7.7
Ger 3 sense 73.3 11.2
Ger 3 antisense 21.0 8.5

Electronic Gel of PCR products

Nothing showed up on the E. Gel. Our imaging machine was not working properly and we could see very little other than blurry ladders. The ladders did not run straight, so there may be problems with our E. Gel. For example, the gel might have been expired.

Purification of PCR products

We used the [Qiagen PCR purification kit with microcentrifuge protocol]. We eluted with 30 μL of Buffer EB to keep the concentration of our end product as high as possible.

Nanodrop of purified genes

See chart in Nanodrop of PCR products for results.

PCR #2 of purified allergen genes

We used 2 μL of purified DNA, leaving around 28 μL of purified DNA products for our digest, ligation, and transformation.

Gel Electrophoresis of PCR #2 products

A gel image of PCR #2 products showed that all lanes (other than the ladder) had DNA segments with lengths of 300bps. This means that purified DNA is contains the correct genes that the initial primers were meant to amplify.