Koeris/Notebook/2007-1-16

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As one finds often, good planning is half the work. Now I need to re-transform both A and B constructs into K12 EMG2 cells in order to make more plasmid, since I can't find the stocks. On the other hand I did find the 06 stock but no plasmid prep... So I got to grow that one up again, too. Bottom line: wasted a day.

Contents

Sequencing of [His]6 constructs

All three constructs need to be sequenced. New sequencing service in use at Boston University: GeneWiz.

Samples need to be dropped off BEFORE 4pm at Larry Valles office LSEB601.

Set up of sequencing

IMPORTANT: DILUTE STOCK PRIMERS to 2pmol/ul

Measure concentration of all 3 constructs

  • 06
  • 07
  • 08

Sample set up guidelines

  • Submit your samples in 0.2 ml, 8-strip PCR tubes, one sample per tube.
  • For the amount of template needed, please refer to the following table.
  • Provide 8 microliters (without primer) or 12 microliters (premixed with primer) of DNA per tube.
DNA type Size (bp) DNA concentration DNA volume in H2O Primer concentration Primer volume
DS plasmid < 6 kb 40-60 ng/µl 8 µl (~500 ng) 2 pmole/µl 4 µl (8 pmol)
DS plasmid 6-10 kb 60-80 ng/µl 8 µl (~600 ng) 2 pmole/µl 4 µl (8 pmol)
DS plasmid > 10 kb 80-100 ng/µl 8 µl (~800 ng) 2 pmole/µl 4 µl (8 pmol)
PCR product < 1 kb 5-15 ng/µl 8 µl (~80 ng) 2 pmole/µl 4 µl (8 pmol)
PCR product 1-2 kb ~20 ng/µl 8 µl (~150 ng) 2 pmole/µl 4 µl (8 pmol)

Label Sample Tubes

Please label your tube on the SIDE with the corresponding sample ID. Cap your 8-strip tubes tightly with matching 8-strip caps. Try to provide some cushion around the tubes to avoid any potential damage during shipping.

Send Samples

Send your samples along with a printed copy of the order summary to GENEWIZ via the preferred overnight courier service at FedEx.

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