User:Lu Wang/Notebook/Team Allergy/2010/07/26

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Procedures

Last week, we finished ligation together Sense and Intron parts of the hpRNA and transformed them into E. coli. This week, we will extract the plasmids, add in the antisense part, and transform.

Procedures today...

  1. Culture colonies (done over the weekend)
  2. Miniprep of V0120 sense + PDK, PAL intron parts
  3. Diagnostic Digest

We also need to check which parts of the sequence on the team page corresponds to the sense/antisense parts of the gene. There may have been mistakes in the past.

Results

Culture We cultured 38 colonies. All grew but one LTP+PAL and Ger+Pal grew. We labeled the cultures 1- 38 for simplicity sake.

1-6: LTP + PDk 7-16: Miniprep

Here are the concentrations of the miniprepped plasmid.

Sample Number
1 2 3 4 5 6 7 8 9 10
LTP+PDK 233.9 507.6 498.7 510.0 485.1 588.3
LTP+PAL 471.8 586.7 500.2 449.4 492.7 430.0 521.8 605.8 499.1 299.9
Bet v1+PDK 430.1 519.3
Bet v1+PAL 432.3 300.6 458.7 213.8
Bet v2+PDK 270.3 223.3
Bet v2+PAL 270.3 223.3
Bet v2+PDK 270.3 223.3
Ger3+PDK 440.4 205.2 342.7 290.2
Ger3+PAL 335.4 297.8 474.3 396.1 354.7 366.2

Diagnostic Digest

We digested the vectors with Spe and Pst1 and ligate with antisense parts (already digested and gel purified). We ran them on the E Gel too long so the lanes do not show anything conclusive, other than that there is digested DNA in the bottom half.

Result:

Ligation of Antisense Part

While another Diagnostic Digest was being set up, we went ahead and ligated all 38 parts with antisense.

Sequence review clarified which parts of the sequences on the team page corresponded to sense/antisense.



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