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oh its a new species? never analyse the dna first?Originally posted by starblue:maybe the primers themselves dun work for those species and those fragments. haiz... genetic stuff are so uncertain. one moment they work for something, then u change a species, it doesnt work anymore. sianzzz... -
/raises eyebrow. that's what the loading dye is for. besides, i'm getting too pro at loading gels now.Originally posted by alexkusu:i mean..maybe u didnt insert them into the wells deep enuf..so float on the top n screw up results ma -
primates. i have 9 species' dna with me now. trying to sequence their MCPH1 gene. currently working on exons 5, 9 and 13.Originally posted by alexkusu:whose dna ru using ah? vero cells? -
i won't even attempt to pretend i know anything...
so, all i can do is hope that you will figure things out soon
good luck sb -
erm, no, not a new species. as in let's say the primers for Exon 5 works for orang utan, then i use it for spider monkey, it may not work. or for howler monkey, the Exon 5 works, but die die also cannot get its Exon 9.Originally posted by hisoka:oh its a new species? never analyse the dna first?
liddat loh. -
Originally posted by starblue:/raises eyebrow. that's what the loading dye is for. besides, i'm getting too pro at loading gels now.
alternatively, just pinch ur classmate finished sample n used the results as ur own -
yeah i meant that its a new species you using the primers/exons onOriginally posted by starblue:erm, no, not a new species. as in let's say the primers for Exon 5 works for orang utan, then i use it for spider monkey, it may not work. or for howler monkey, the Exon 5 works, but die die also cannot get its Exon 9.
liddat loh.
btw so you now everydya busy in uni arh? -
how did it go with exon 13?Originally posted by starblue:primates. i have 9 species' dna with me now. trying to sequence their MCPH1 gene. currently working on exons 5, 9 and 13. -
not a matter of figuring out things. it's more like trial and error. cos we're working with primers and enzymes, and they may just not respond cos the temperature differs by 1 degree, or cos the dna differs by 1 base.Originally posted by the Bear:i won't even attempt to pretend i know anything...
so, all i can do is hope that you will figure things out soon
good luck sb
even the seniors in my lab also can only advice me on what other stuff i can try, but they also cannot predict what i will get. one lady even said that if i get too desperate (like how she was last time), i can try to do something utterly counter-intuitive. like the usual method is to lower the annealing temp by 2 degrees each time it fails. instead, maybe can increase by 1 degree. that sort of thing. lolz..
it just gets on your nerves when nothin works after 6 attempts. -
wad u mean how did it go? some species i got the exon 13 already.Originally posted by alexkusu:how did it go with exon 13? -
it's a urops project. i'm the only one in it. there is no classmate's results to pinch from.Originally posted by alexkusu:
alternatively, just pinch ur classmate finished sample n used the results as ur own -
yah gotOriginally posted by alexkusu:how come so good
death got class tmrw? -
yeah... everyday stay as long as i can in the lab to run all those PCRs and gels and dna sequencing. i plan to stay up till 8pm everyday on days i dun have tuitions.Originally posted by hisoka:yeah i meant that its a new species you using the primers/exons on
btw so you now everydya busy in uni arh? -
orh.. (i'm totally lost)
Originally posted by starblue:not a matter of figuring out things. it's more like trial and error. cos we're working with primers and enzymes, and they may just not respond cos the temperature differs by 1 degree, or cos the dna differs by 1 base.
even the seniors in my lab also can only advice me on what other stuff i can try, but they also cannot predict what i will get. one lady even said that if i get too desperate (like how she was last time), i can try to do something utterly counter-intuitive. like the usual method is to lower the annealing temp by 2 degrees each time it fails. instead, maybe can increase by 1 degree. that sort of thing. lolz..
it just gets on your nerves when nothin works after 6 attempts. -
wat ru havin prob with leh? exon9?Originally posted by starblue:wad u mean how did it go? some species i got the exon 13 already.
maybe its a dummy gene ur lecturer threw in to piss u off
u knw..they wana see u record down observations/results of 'failed' experiments -
er.... suffice to say that it is hard to troubleshoot if a PCR fails. cos there are many things contributing to success. so u always have to trial and error-ly try out every variation you can and see what you get.Originally posted by the Bear:orh.. (i'm totally lost) -
she has a point there leh.. 6 attempts NIA!Originally posted by starblue:not a matter of figuring out things. it's more like trial and error. cos we're working with primers and enzymes, and they may just not respond cos the temperature differs by 1 degree, or cos the dna differs by 1 base.
even the seniors in my lab also can only advice me on what other stuff i can try, but they also cannot predict what i will get. one lady even said that if i get too desperate (like how she was last time), i can try to do something utterly counter-intuitive. like the usual method is to lower the annealing temp by 2 degrees each time it fails. instead, maybe can increase by 1 degree. that sort of thing. lolz..
it just gets on your nerves when nothin works after 6 attempts.
how many machines u have in the lab? if possible, use all available machines so dun have to wait in between the attempts for results -
erm... nope. all 3 fragments worked for at least 1 species. but you know lah... primers you order from the companies are usually "general" primers, so may not fit all species, even though they should theoretically.Originally posted by alexkusu:wat ru havin prob with leh? exon9?
maybe its a dummy gene ur lecturer threw in to piss u off
u knw..they wana see u record down observations/results of 'failed' experiments -
so sad.Originally posted by starblue:yeah... everyday stay as long as i can in the lab to run all those PCRs and gels and dna sequencing. i plan to stay up till 8pm everyday on days i dun have tuitions.
want to have dinner some day when ou stay back? -
hello starblue mei!
i cant stay long here cos i m abt to head out already, and tonite is the first time i m driving home alone w/o my parents beside me.
pray for my safety peeps -
6 attempts is usually the max. every attempt you do, you drop annealing temp by 2 degrees. so the protocol prediction says the primer should use 56 degrees celcius. at the 6th attempt, you're using 44 degrees, (which is VERY off the "optimum" already) and anything lower than that there is no point even trying.Originally posted by alexkusu:she has a point there leh.. 6 attempts NIA!
how many machines u have in the lab? if possible, use all available machines so dun have to wait in between the attempts for results
got 5 machines. but then have to let other people in the lab use also mah... cannot just book for myself wad. plus each run takes 3 hours. a lot of waiting time in between loh. -
from my experience the labs in nus run til pretty late and have alot of ppleOriginally posted by alexkusu:she has a point there leh.. 6 attempts NIA!
how many machines u have in the lab? if possible, use all available machines so dun have to wait in between the attempts for results -
given that everythin is right...or maybe the samples u received has degraded sufficiently to give poor results?Originally posted by starblue:erm... nope. all 3 fragments worked for at least 1 species. but you know lah... primers you order from the companies are usually "general" primers, so may not fit all species, even though they should theoretically.
or maybe its human error
do you have anything to benchmark ur results on? -
muakzz rainee jie!! so long never come in to see you le.Originally posted by rainee:hello starblue mei!
i cant stay long here cos i m abt to head out already, and tonite is the first time i m driving home alone w/o my parents beside me.
pray for my safety peeps
drive carefully hor... now so dark already very dangerous leh... -
rainee, miscall me when u get back home safe