PCR failure. GC problem? - (Jan/09/2008 )
I am trying to amplify this region of DNA (relA gene) with a GC% of approximately 65.6% from bacterial genomic DNA.
i used.
1ul Forward Pri@ 5uM(stock conc.) Tm= 58.4
1ul Reverse Pri @5uM (stock conc.) Tm = 64.5
1ul Genomic DNA (6ng/ul)
1ul dNTP (10mM)
1ul of Taq ( 1.5 U/ul)
5ul of 10X PCR buffer
40ul of H20
Initial denaturation @ 95 for 3min.
25 Cycle of
95 for 30sec
52 for 30sec
72 for 3 min ( that guy is about 3kb)
then
30 min of 72 ( TA cloning purpose)
NO band at all @.@. BUt positive control using another primer pair that amplify the 16sRNA turns out on gel.
the GC content of 16sRNA ( i sequenced it before = 52.66%)
Who thinks that's some significant difference, holler back!
I am going to do a hot start for 10 min. Not quite planning to add "drug" into the PCR mixture just yet.
Any suggestion will be appreciated!
I guess by "drug" you meant additives? Anyway I'd strongly recommend you do, for high GC content if you don't want to increase temperatures try this:
The 5x concentrated Combinatorial Enhancer Solution (CES) contains:
2.7 M betaine
6.7 mM DTT
6.7% DMSO and
55 μg/ml BSA
http://www.protocol-online.org/prot/Protoc...lates-4054.html
Good Luck!
I see several things that should be fix:
1. very low anneling temp for the primers specialy for a high GC% (if primers are 58C and 64C the anneling should be in between more or less, can begin with 61C and increase or decrese 2C as needed).
2. Too few template add more dna at least 50ng then adjust quantity if you see too much in the gel.
3. Too short cycles for 3Kb amplification you are not given enouhg time to the chains to open properly. I sugest 95C 1min, 61C 1min, 72C 2-3 min use at least 35 cycles and add a hot start 95C 6-8 min
4. Try in 25 µL reaction to check if you have it correct, then move to 50µL reaction so you save materials that are expensive.
Note:
There are Hot start Taq that have special buffers for high GC if you continue with problems can use this type of enzyme
1. very low anneling temp for the primers specialy for a high GC% (if primers are 58C and 64C the anneling should be in between more or less, can begin with 61C and increase or decrese 2C as needed).
2. Too few template add more dna at least 50ng then adjust quantity if you see too much in the gel.
3. Too short cycles for 3Kb amplification you are not given enouhg time to the chains to open properly. I sugest 95C 1min, 61C 1min, 72C 2-3 min use at least 35 cycles and add a hot start 95C 6-8 min
4. Try in 25 µL reaction to check if you have it correct, then move to 50µL reaction so you save materials that are expensive.
Note:
There are Hot start Taq that have special buffers for high GC if you continue with problems can use this type of enzyme
Alright sounds like a plan to me. mind if i ask some questions,,
1. Do you know why we need high annealing temperature for this particular reaction? I used to think it shouod be 5 -10 below.
2. From the book ( qiagen), it says 10pg to 10ng of DNA. can i try with the max of 10ng instead?
I would increase the length of the primers to give a Tm ~70, and adjust the annealing temp to ~60-62 degrees. Taq pol isn't very active below 65 C, and your primers (especially the forward primer) will have fallen off the template by then. The GC content isn't too bad, so I would adjust the rest of the reaction before worrying about additives.
Thanks for everything. i modified the denaturatin time to 1 min and longer annealing time 45 sec. and a hot start of 8 min. saw a clear nice band at 3kb.
The anneling temp should be near the Tm temp so the primers can attach properly to the chain. What I do if diffent temp for a primer set just use the averagebetwen them, then adjust if necessary. For example the average of your primer set is 61C if got several bands plus the interest one then you increase temp increments of 2C until got one band. If no band then decrease temp.
Other imp. thing is that the chain must open completly so the primers can attach in the proper place. You have a big amplicon so need more time for this.
about quantity you can use between 10pg-100ng it depends the nature of the sample and many other things. You told that no band so I'm just telling to go a little bit extreme then adjust the parameters until get a good result. For PCR not always following the book will get you a result, sometime must change things until a results is see. So be patient and pray to the gods of PCR for a good band
Other imp. thing is that the chain must open completly so the primers can attach in the proper place. You have a big amplicon so need more time for this.
about quantity you can use between 10pg-100ng it depends the nature of the sample and many other things. You told that no band so I'm just telling to go a little bit extreme then adjust the parameters until get a good result. For PCR not always following the book will get you a result, sometime must change things until a results is see. So be patient and pray to the gods of PCR
for a good bandOH my god... I ran another PCR from the PCR mixture and got multiple bands i think i need to do a gradient now or maybe purify the sample or both.
@.@
And i thoguht it's better to amplify from an already PCR product who knows this thing came out hahaha