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Curtis

Member Since 30 Jan 2009
Offline Last Active May 22 2012 08:37 PM

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Topics I've Started

Desired length of overlap region for OE-PCR

09 April 2012 - 10:02 PM

I am designing primers to produce two PCR products that have an overlapping region, and then run Overlapping-Extension-PCR with these PCR products as template.

Some people say keep the overlapping size at 40-45 bp, but this way the annealing temperature will be very high (nearly 81C). I want to keep it at 21 bp, meaning desining two primers that are exactly complementary of each other (Reverse primer of Fragment 1 to be complementary to Forward primer or fragment 2). should I worry about the length at all? or they will bind anyway?

How to replace all hard returns in Microsoft Word?

25 March 2012 - 07:48 PM

Sometime I have to copy/paste genome sequences from NCBI to Microsoft Word, and as you know all sequences come with line numbers on left side and space breaks for every 10 bp. Now, it is easy to replace 'any digit' or 'white space' in Word, but how to replace all hard returns (enter)? I can't find any option!

I need these sequences to add to Mega 5 alignment software, and it only accepts sequences with no breaks in between.

Does 16s rRNA gene prove all Abrahamic religions wrong?

15 March 2012 - 08:37 PM

I am agnostic, never believed in any religion or faith. If according to Abrahamic books there were an Adam and Eve, made from clay by God, then how do religious scientists explain 16s rRNA gene. I can't consider anyone as scientist if they are religious.

I think there is already enough proof in the world for intelligent people. It's just that some brains can't still handle the reality.

I read somewhere that 60% of American scientists are agnostic.

Is it only water and nucleic acid in the top phase?

15 March 2012 - 08:41 AM

After treating cDNA with RNase H I did a phenol/chloroform extraction to purify my cDNA, however I forgot to add isopropanol to pellet down the cDNA, and I used the top phase directly in PCR and surprisingly I got PCR product.

what is the component of top phase during phenol/chol extraction? is it just water and nucleic acid??

why Phusion polymerase is not recommended for overlap PCR?

15 February 2012 - 08:06 AM

According to this protocol it is not recommended to use Phusion polymerase in overlap PCR reactions (4.2), Why?

http://openwetware.o...erlap_Extension