User:Lu Wang/Notebook/Team Allergy/2010/06/22

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Procedures

Today's objective is to take our PCR products from yesterday (LTP sense, Ger 3 sense, and Ger 3 antisense)and insert them into vector V0120 for E. coli.

Our procedures were:

1. Gel Extraction of LTP sense, Ger 3 sense, and Ger 3 antisense 2. Purification of LTP sense, Ger 3 sense, and Ger 3 antisense 3. Digest of LTP sense, Ger 3 sense, and Ger 3 antisense with Xba and Pst restriction enzymes 4. Purification of digested allergens LTP sense, Ger 3 sense, and Ger 3 antisense

5. Digest of V0120 with Xba and Pst restriction enzymes 6. Gel Electrophoresis of the digested vector 7. Gel Extraction and Purification of Vector

8. Ligation of V0120 and inserts for each LTP sense, Ger 3 sense, and Ger 3 antisense

Results

1. Gel Extraction of LTP sense, Ger 3 sense, and Ger 3 antisense 2. Purification of LTP sense, Ger 3 sense, and Ger 3 antisense

Obtained 2.6, 7.8 and 7.9 ng/μL of LTP sense, Ger 3 sense, and Ger 3 antisense, respectively, after ourgGel extraction and purification. These are relatively small concentrations. These tubes of DNA will be digested and then purified again.

3. Digest of LTP sense, Ger 3 sense, and Ger 3 antisense with Xba and Pst restriction enzyme

4. Purification of digested allergens LTP sense, Ger 3 sense, and Ger 3 antisense

Obtained 1.5, 4.3 and 0.5 ng/μL of digested LTP sense, Ger 3 sense, and Ger 3 antisense, respectively. The drop in concentration is attributed to loss of DNA through the purification process.

5. Digest of V0120 with Xba and Pst restriction enzymes 6. Gel Electrophoresis of the digested vector

The properly digested vector is 3000bps long, and rests between two bands on the gel -- the uncut or one cut vector and the removed inserts.

7. Gel Extraction and Purification of Vector Nanodrop of digested vector yielded 25.2 ng/μL.

8. Ligation of V0120 and inserts for each LTP sense, Ger 3 sense, and Ger 3 antisense

Ligated vectors were put on ice and set aside for tomorrow, where they will be transformed into competent turbo E. coli, which will be grown in plates of agar. Colonies that grow should contain closed vector plasmid, which contains ampicillin resistance and inserts.


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