Title: PCR of subjects' DNA (24 for Chinese, Malays and Indians each)
1. Prepare master mix for 75 samples containing the following components:
2. Pipette 8ul of the master mix into each well of the 96-well PCR plate.
*those boxes coloured are filled with the 8ul master mix.
3. Pipette 2ul of the DNA into the wells in the following order:
4. Cover the plate with silicon mat
5. Spin down the plate at 200G for 20 sec
6. Put the plate in the PCR machines. The standard conditions for PCR are:
7. After PCR has finished, spin down the plates at 200G for 20 sec
That's all for now...hope u guys enjoy the last few weeks of your SIP...all da best guys!!!!:)
Nur Zahirah
0503165C
TG02
5 comments:
Hey
Just wondering, you have 72 samples yet you prepared master mix for 75 samples. Me and my group mates do this too because strangely enough, there usually is not be enough for the last tube. Im just wondering if you know why.
Another question, why don't you gals use a control?
Thanks
Azhar
Hey
What do u need to cover the plate with silicon mat? What's the use the silicon mat See u =)
Eugene Wong
TG02
heya azhar,
to answer your first qn....may i ask if u use a multi-channel pipette or a single channel pipette??...usually if multi-channel pipette is used, we would have just enough for 72 samples...but then i usually dun use them because i dun trust them..haha...now here's what i think happen along the way
1)there can always be error in pipetting when pipetting the liquid out and when dispensing them i.e. the solution pipetted is lesser than the amount it should be or not all was dispensed inside the master mix resulting in lesser vol.
2)the pipettes are not accurate. this happens in my lab. some of the pipettes, perhaps use too many times until it overwork n decided to break down..sometimes we might not realise that until sometime later
3)when u prepare the master mix..you would vortex it to mix well....not sure if you'll spin it down after that but we dun...so when you open the master mix tube, some minute amount of liquid at the underside of the 'cap' or near it can 'splash' out so you have lesser amount
4)no matter how good and accurate your pipetting skills are, when dispensing the master mix, 1/10 of the master mix will be gone. this could also be one of the reason why using single channel pipette results in insufficient master mix but not multi-channel..because single channel you'll use the same pipette tip and have to dispense into 72 wells compared to multi-channel they only dispense tt 1 row....so obviously more will be gone for single channel
tt's the possible reasons i could come up wif for now..hope it explains....for your 2nd qn...i noe that it's gd to run controls...even notes given to us got state we must run cntrols containing different components..if i still remember no DNA..bystander DNA..etc...however, each PCR tube cost $5 and we are running large amt of samples...(300 altogether) and 45 fragments of the gene...if we were to run controls...you can imagine how much money we are gonna waste n i think my supervisor will juz scream at us...soo what we do is after running PCR..we do something called the checking gel...the gel is the same as wat i posted for gel electrophoresis last time but the wells used are much smaller due to lesser amt loaded...this is used to check if PCR has occurred i.e has a lot of strand of that fragment has been synthesized wonder how we know it's the fragment we 1..they could always PCR other parts of the gene....well...firstly the primers are designed by my officers such that they anneal to the specific location...and to know if really the PCR occurs at that fragment, well we have to wait for DNA sequencing results...bcoz we are using a software called seqscape to analyse the sequence aka blasting...this software requires us to put in the reference/the sequence for that fragment that we are doing...soo if we get unassembled or reverse orientation in that software, it means that either wrong primers were used or perhaps primers might anneal wrongly...but usually it's not the latter........
heya eugene
well the silicon mats is used to cover the plates to prevent the solutions in the tubes/plates from evaporating...PCR uses the elements of heating and cooling and thus if we do not cover it with silicon mats it would evaporate and at the end of waiting soo long for it to finish, you'll find there's nuthing or very little amount of PCR product in the tubes...you can use plastic...but then you can never trust what the manufacturer write..coz we got use this different plastic cover..ran out of silicon mat and the normal plastic we used...and it was written PCR compatible but in the end it melted and the volume of our PCR products was lesser i.e. some evaporated. so the best is silicon mats.....
to azhar.....
ermm...the $5 i write is tubes + the PCR components...tubes only does not cost tt expensive...:D
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