Fragment sizes of H-Lo ladder

Gel 1: Pilar & Jim High Molecular Weight DNA (extraction) PCR product That’s how it ideally should look like: nice single band Here there is some HMW DNA, but also a lot of degraded DNA. This probably would still be ok for PCR Probably overloaded (too much DNA). Could also be too much template DNA in the PCR L Origin (wells) This is RNA HMW DNA (top) 1 (Pilar)-Sockeye #10-Salt 2 (Pilar)-Coho #2-Salt 3 (Pilar)-Sockey #10-Qiagen 4(Pilar)-Coho #2-Qiagen L-Ladder 5-(Jim)-Sockeye #10-Salt 6-(Jim)-Chinook #4-Salt 7-(Jim)-Sockeye #10-Qiagen 8-(Jim)-Chinook #4-Qiagen PCR product (bottom) 1-(Jim)-Chinook #4-Salt 2-(jim)-Chinook #4-Qiagen 3(Jim)-Sockeye #10-Salt 4-(Jim)-Sockeye #10-Qiagen L-Ladder 5-(Pilar)-Sockeye #10-Qiagen 6-(Pilar)-Coho #2-Qiagen 7-(Pilar)-Sockeye #10-Salt 8-Negative control Conclusion: No obvious effect of salt vs Qiagen extraction, either in HMW or PCR product

L Good HMW DNA with little degradation, but the PCR didn’t work well Gel 2: Claudia, Kristi & Nick

Gel 3: Brian & Gang Weak HMW DNA with some degradation, but the PCR worked well in three samples (all Quiagen extractions!) L L PCR product L: ladder 1: Sockeye 7 salt (GX) 2: Coho 3 salt (BK) 3: Sockeye 8 Quiagen (BK) 4: Coho 3 Quiagen (BK) 5: DNA A Quiagen (GX) 6: DNA B Quagen (GX) 7: Chinook 2 salt (GX) 8: control HMW DNA L: ladder 1: DNA A Quiagen (GX) 2: DNA B Quaigen (GX) 3: Chinook 2 salt (GX) 4: Sockeye 7 salt (GX) 5: Coho 3 salt (BK) 6: sockeye 8 salt (BK) 7: Coho 3 Quiagen (BK) 8: Sockeye 8 Quiagen (BK)

Gel 4: Alfred and Danny Good HMW DNA in all samples (#8 a bit weak) Good PCR products in 5 of 7 samples This is probably template DNA, i.e. there was too much in the PCR reaction – it still worked though RNA Degraded DNA PCR product HMW DNA 1Sockeye 1 (Salt) 1Hi-Lo Ladder 2Nothing 2Sockeye 1 (Salt) 3Steelhead 1 (Salt) 3 4 Sockeye 1 (Qiagen) 4 5Sockeye 4 (Salt) 5 6Chinook 3 (Salt) 6 7 Sockeye 4 (Qiagen) 7 8Chinook 3 (Qiagen) 8 9Control 9 Steelhead 1 (Qiagen) 10Hi-Lo Ladder 10

DNA looks ok in 4 samples Gel 4: Bill & Thomas Gel broke to two pieces PCR worked fine as well – this is actually the gel where we saw most straight away. Something strange going on here – you can’t see it on the picture, but on the gel the stain was only on the bottom surface of the gel – a lesson that you can’t see everything on a picture. DNA 1.Chinook 1 salt 2.Chinook 1 Qiagen 3. Sockeye 6 Qiagen 4. Steelhead 3 Qiagen 5. Sockeye 9 Qiagen 6. Steelhead 3 salt 7. Sockeye 9 salt 8. Ladder PCR product Ladder 1. Sockeye 9 salt 2.Steelhead 3 salt 3. Sockeye 9 Qiagen 4. Steelhead 3 Qiagen 5. Sockeye 6 Qiagen 6. Chinook 1 Qiagen 7. Negative control 8. Chinook 1 salt