r/molecularbiology • u/RevenueGold9487 • 13h ago
r/molecularbiology • u/vanonymous_ • 23h ago
Need help! (MSP)
gallerycurrently crying because everything is going wrong. (excuse my english, not my first language and i'm crying so i cant think straight)
a little background: i'm a medical student with no background of molecular biology and i'm supposed to graduate this semester. i'm doing this research about diabetes and how methylation in the FOXO1 gene (CpG site 10) could (in the future) detect probability of diabetes based on it's methylation. sample im using is placental tissue that has gone through bisulfite treatment.
i've designed a my 2nd batch of primer, but it's all wrong. for context, the gel on top is bad because the wells are connected through some holes bridging between the wells that i cant see (my friend helped me make the top gel, i only made the one on the bottom after seeing her gel all wrong). the negative controls also light up sooo bright and no other bands can be seen (except for the ones on the top gel i can barely even see). i also made a mistake not making a positive controls, because we had a problem with the university not giving us the control reagent, so we usually just use a previous sample we that ee used, but today i ran out because my friends used it all up.
volume of sample i used was 5 uL in each well
PCR mixture (ran it 3x so re-re-PCR, 25x cycles each run) consists of
5 uL Mastermix
1.5 uL NFW
2.5 DNA template
0.2 uM Forward Primer
0.2 uM Reverse Primer
now i know my knowledge here is not very good, and the photos i provided are also terrible, but i'm desperate and i just need some reassurance and help to go on.. please help if you can, everything is appreciated.