Koeris/Notebook/2007-1-16
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.
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.
