| x |
|
|
− |
+ |
+ |
+ |
|
x |
|
− |
− |
− |
+/− |
| x |
x |
|
− |
+ |
− |
+/− |
|
|
x |
− |
− |
− |
+/− |
| Interpretation |
Bad mastermix/wrong settings/faulty amplifier |
Not enough template |
Lysed yeast inhibited PCR |
Uneven template load/digestion |
| Recommendation |
Double-check/repeat |
Add more template/improve lysis |
Add less template |
Adjust template load/re-PCR from obtained bands |
|
| Double bands may NOT be observed on the PCR done on the mixture of the plasmid prep with yeast, because smaller product from
the empty vector may out-compete the bigger one from the plasmid.
|
| aJG4-5-Raf1 is a suitable template; be sure to use a control library plasmid (with the insert) of the same type as is in the
library.
|
| bPCR from the empty vector yields a product of ~130 bp for JG4-5 (FP1 and FP2 primers); ~185 bp for YesTrp (YesTrp forward
and reverse primers).
|
