0
7 replies to this topic
#1
epicrazy
-
- Active Members
-
- 32 posts
Enthusiast
0
Neutral
Neutral
Posted 06 May 2010 - 03:13 AM
Having gone through days of many PCRs a day, I realised the damage of freeze thawing primers and dNTPS! I would recommend to anyone doing multilple PCRs a day, to use a fresh (or not recently thawed) aliquot of primer and dNTP for each run. WHen you make up dNTPs and primers, aliquot them to several tubes (and each time you use a tube, mark it so you know how many times you have thawed them).This way, you dont use one aliquot over several days, and rotate between aliquots. For some reason if I used the same tube consecutively for a few days, sometimes it worked and sometimes it didnt leaving me puzzled with a blank gel! Just thought it might useful for beginners at PCR!
#2
Trof
-
- Global Moderators
-
- 946 posts
Brain on a stick
66
Excellent
Excellent
Posted 06 May 2010 - 06:04 AM
If you run more PCRs in a day then you should let the primers and stuff on ice and just don't freeze it again. Freeze-thaw cycles make more damage than leaving it on ice for some time.
I leave my primers in the fridge when I'm using them every day.
I leave my primers in the fridge when I'm using them every day.
Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.
I never trust anything that can't be doubted.
I never trust anything that can't be doubted.
#3
epicrazy
-
- Active Members
-
- 32 posts
Enthusiast
0
Neutral
Neutral
Posted 06 May 2010 - 06:28 AM
I have tried leaving it on ice or fridge the whole day and freezing them at the end of the day. I havent tried leaving them in the fridge for a few days because I wasnt sure how the dNTPS would handle it
#4
PandaCreamPuff
-
- Active Members
-
- 43 posts
Enthusiast
0
Neutral
Neutral
Posted 10 May 2010 - 05:48 AM
This may sound crazy but, besides Taq, I put all my working stocks required for PCR (primers dNTPs MGCL2 10x buffer) in a 4C fridge. I had been doing this for months and I have had no problems with my PCR.
#5
epicrazy
-
- Active Members
-
- 32 posts
Enthusiast
0
Neutral
Neutral
Posted 10 May 2010 - 07:31 AM
That s great for you! For me, it varies between different primer sets... some seem to be more sensitive than the others wrt freeze/thaw... so i uniformly store them at -20
#6
Adrian K
-
- Global Moderators
-
- 684 posts
Legendary Graduate Beggar
24
Excellent
Excellent
Posted 10 May 2010 - 09:37 AM
For me, i do a master mix and aliquote into each individual tube and frozen it. another master mix which consist of h20 and taq will only be mixed before i run my pcr, and will add together with template into the frozen tube to "melt" it.
So far, no problem for my multiplexes.
So far, no problem for my multiplexes.
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.
..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...
"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong
"It's all just DNA. Do it."---phage434
..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...
"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong
"It's all just DNA. Do it."---phage434
#7
Maddie
-
- Active Members
-
- 289 posts
Veteran
2
Neutral
Neutral
Posted 12 May 2010 - 05:35 AM
Adrian, have you created a new way to make a hotstart? 
Theory is when we know everything and nothing is working. Practice is when everything is working and nobody knows why. Here, we combine theory and practice. Nothing is working and nobody knows why.
A. Einstein
A. Einstein
#8
Adrian K
-
- Global Moderators
-
- 684 posts
Legendary Graduate Beggar
24
Excellent
Excellent
Posted 19 May 2010 - 02:03 AM
Maddie, on May 12 2010, 09:35 PM, said:
Adrian, have you created a new way to make a hotstart?
Huh?....did I...?
But one thing for sure, I'm using hotstart Taq.
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.
..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...
"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong
"It's all just DNA. Do it."---phage434
..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...
"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong
"It's all just DNA. Do it."---phage434
