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