1. Run the hybridization PCR samples on 1.5% agarose gel and EL400 gel.
2. Repeated mixed PCR products (form Volkan) hybridization with fresh hybridization buffer:
New 20 x SSC buffer (store) and 10% SDS stock (Chunyang) have been used to make up fresh hybridization buffer (2x, 1x). New 6x SSC buffer and 4X SSC buffer have been made.
Finding: at the beginning, SDS (Volkan) could not be dissolved in the buffer (1x Hyb and 2x Hyb) except in 45degree water bath. But Chunyang’s 10% SDS stock solution can be dissolved in the buffer. The concentration of the previous SDS stock may have problem and affected the hybridization result.
5 ul mixed PCR product
20 ul 2uM biotinated primers
30ul 2x hybridization buffer
Run PCR on machine 17 overnight.
3. Sowed lettuce seeds
1. Placed the hybridization + Dynabean samples in the incubator shake for 1.5 hour in 45 degree.
2. Processed the washing procedure then eluted by EB buffer stored in -20 degree.
3. Sequence result analysed: although the results are empty insertion, it worth to use EcoR I to digest the DNA to check the result. And re do the sequence by using M13 forward primer.
Sample 1: microsatellite region is reported when blast the first 1-320 bases.
To do list:
. Run PCR for the new hybridization samples
. EcoRI digest minipre product
. Re run the sequence for cloning sample by using M13 forward primer
. CE facility key
. Inoculation/ seed sowing/new culture system for CE test