Sequencing of Difficult template
For constructs cloned into vectors such as pT7A3, pT7A30
Protocol 1
[edit]1. Transfer >270 ng plasmid solution to a microcentrifuge tube or PCR Tube.
2. Evaporate H2O to almost dryness.
3. Add 4 ul of 5X PCR Enhancer Mix. by pipetting.
4. Add 1.5 ul 10 uM sequencing primer, pT7A3-seq-F.
Protocol 2
[edit]1. Grow E coli in 15 ~30 ml of 2XYT or SB medium containing antibiotics, e.g., K, at 37 oC o/n, in a shaker incubator.
2. Spin down cells
3. Pour off medium
4. Add 0.5 ml P1, suspend cells
5. Add 0.5 ml P2, mix well
6, Add 0.7 ml N3, votex
7. Transfer to 2 ml tube, spin 2 X 2.5 min at 13000 rpm
8. Transfer supernatant to a spin column
9. Wash with 0.5 ml PB, then 0.75ml PE
10. Spin 1 min
11. Elute DNA with 20 ul EB into a new tube
12. Elute DNA with another 50 ul EB into another tube.
13. Measure DNA concentration with nanodrop
14. If DNA concentration is less than 250 ng/ul, concentrate the DNA solution by heating on 50 oC block for 30 min.
15. Add 2 ul of 250 ng/ul DNA to PCR tube, Add 4 ul 5 X PCR enhancer mix, 1.5 ul 10 uM sequencing primer.
For pT7A3 or pT7A30 vectors: use pT7A3-seq-F sequencing primer
16. Order sequencing on genewiz.com, use premix. Do not use difficult template because it costs $8