User:Anthony Salvagno/Notebook/Research/2009/03/17/D&L Day 4

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Today, so far and to do:

  • P/C extraction of overnight digestion
  • EtOH precipitation
  • Make minigel
  • Run minigel comparing digestion with uncut
  • If digestion worked well, we will do the ligation.

Gel Results and Remarks

Ligation Attempt

Gel Prep
Tube Number Vol plasmid DNA Vol gDNA Vol Buffer 10x Vol T4 DNA Ligase Vol H2O Total Vol
1 6.7ul ---- 2uL 1.5uL 9.8uL 20uL
2 6.7uL 2uL 2uL 1.5uL 7.8uL 20uL
3 3.3uL 2uL 2uL 1.5uL 11.2uL 20uL
4 1.3uL 2uL 2uL 1.5uL 13.2uL 20uL
5 1.3uL 5uL 2uL 1.5uL 10.2uL 20uL
6 1.3uL 10uL 2uL 1.5uL 5.2uL 20uL
  • Allow to incubate at 16C overnight (in PCR chamber thing).

This time the ratios, by weight, are (V:I, vector:insert):

  1. V only
  2. 5:1
  3. 2.5:1
  4. 1:1
  5. 1:2.5
  6. 1:5

Digesting Another Try

Gel Prep
Tube Number Vol gDNA Vol and Type 10x Buffer Vol BSA 10x Vol XhoI Vol H2O Total Vol
1 6.4uL NE2/7.5uL 7.5uL 3.5uL 46.6uL 75uL
2 12.8uL NE2/7.5uL 7.5uL 3.5uL 40.2uL 75uL
  • Add 3.5uL more XhoI after ~2 hours

Mary Ann told Kelly and I we might get better digestion if we use less DNA, so we lowered the amount in the sample.