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badguy's Content

There have been 19 items by badguy (Search limited from 16-April 20)


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#156996 Problem with 3 step PCR

Posted by badguy on 27 June 2013 - 12:39 AM in PCR, RT-PCR and Real-Time PCR

i tried your method before and ended up getting a lot of unspecific bands.
i ended up using the old method, which is multiple cloning.



#156941 ARMS-PCR

Posted by badguy on 25 June 2013 - 08:29 PM in PCR, RT-PCR and Real-Time PCR

I'm having some trouble when using single cell ARMS-PCR for Beta thalassemia HbS cd6 A>T.

After the PCR, mutant band was observed in normal sample. any suggestion to improve the specificity of primers?

PCR Master mix-final volume 20ul
5x Bioline MyTaq Buffer 4ul
primer final conc. 0.2uM
Taq 2.5U

Annealing temperature at 65C

below is the primer sequence.

HbS-Wildtype 5'- AAC AGA CAC CAT GGT GCA TCT GAC TCC TGA -3'
HbS-Mutant 5'- CCC CAC AGG GCA GTA ACG GCA GAC TTC TCC A -3'
A primer 5'- CCC CTT CCT ATG ACA TGA ACT TAA -3'
B primer 5'- ACC TCA CCC TGT GGA GCC AC -3'
A-out primer 5'- GAG ACT TCC ACA CTG ATG CAA TC-3'
B-out primer 5'- GAA GTC CAA CTC CTA AGC CAG T-3'

Below is the reference I used.
http://www.ithanet.e...on_system_(ARMS)
1st PCR was run using HbS-Wildtype,HbS-Mutant, A-out and B-out primer.
2nd PCR was run using HbS-Wildtype,HbS-Mutant, A and B-primer.

I also tried touchdown pcr, which is Ta-70C for first cycle then minus 0.5C for 10cycles, final Ta-65C for 20 cycles. The mutant band still can be observed after the PCR.



#153174 PCR with Plasmid recovered from filter paper

Posted by badguy on 01 April 2013 - 04:48 PM in PCR, RT-PCR and Real-Time PCR

If that is the case, just elute the plasmid with 50ul of TE buffer or less by suspending and centrifuging at full speed if my memory serves me right.
after that just add in your competent cells and do the transformation.



#153091 Annealing temperature for PCR

Posted by badguy on 27 March 2013 - 11:28 PM in PCR, RT-PCR and Real-Time PCR

The formula I used is Ta= 2(A+T)+4(G+C) - 2
I was taught to use that formula and so far so good.



#153090 PCR with Plasmid recovered from filter paper

Posted by badguy on 27 March 2013 - 11:25 PM in PCR, RT-PCR and Real-Time PCR

you can use 50ul of TE buffer/nuclease free water to elute the plasmid.
It's better to do transformation rather than doing PCR directly. You never how much of plasmid has been loaded into the paper.
Is the plasmid in FTA card? There are instructions on their website on how to elute plasmid from it.



#147836 DNA polymerase for GC rich template?

Posted by badguy on 10 January 2013 - 06:49 PM in PCR Reagents and Equipments

you can't really say. I'm doing some difficult pcr with Finnzyme's Phusion which comes with a GC buffer only for GC rich sequeces. We used Bioline's, it didn't amplify our fragment

What Bioline's polymerase you used?



#147734 DNA polymerase for GC rich template?

Posted by badguy on 08 January 2013 - 11:55 PM in PCR Reagents and Equipments

In among all the brands of DNA polymerase, which one stand out among all?
I found out Bioline's MyTaq look pretty good when compare to other vendors' DNA polymerase with DNA template of 61%GC.
Anyone uses Bioline's product before?



#147096 Thermal cycler recommendation

Posted by badguy on 23 December 2012 - 09:48 PM in PCR Reagents and Equipments

I have come across this thermal cycler: Speedcycler2, which heating rate of 15C/s and cooing rate of 10C/s.
So far I only see heating rate of 4C/s and cooling rate of 3C/s in most thermal cyclers.



#146881 Thermal cycler recommendation

Posted by badguy on 17 December 2012 - 09:30 PM in PCR Reagents and Equipments

Any good thermal cycler to recommended?
currently i laid my eye on Biorad's C-1000. any other better than this?



#146880 Best proof-reading polymerase?

Posted by badguy on 17 December 2012 - 09:28 PM in PCR Reagents and Equipments

I read from Biotechniques, it says that Phusion from Finnzymes has the best proof reading activity.



#143772 deletion of large fregments

Posted by badguy on 19 October 2012 - 07:37 PM in Molecular Cloning

Your gene of interest is cloned into a plasmid right? I think you can try inverse PCR.



#143078 Primers Freeze-Thaw

Posted by badguy on 10 October 2012 - 01:27 AM in Molecular Biology

i always keep my working stock in -20C. so far so good. after 1-2 years the primers stil working great



#143061 Storing freshly autoclaved LB agar solution?

Posted by badguy on 09 October 2012 - 08:03 PM in Molecular Biology

My lab's practice is do not keep the autoclave agar in the oven for not more than 24 hours, and mix well before keep inside the oven as agar will tend to set at the bottom.



#143057 Fastest way to detect bacteria?

Posted by badguy on 09 October 2012 - 06:40 PM in Microbiology

you still need to do sequencing after the 16s rRNA PCR to identify the bacteria, which also needs a few days to get the result.
maybe can use biochemical test kitPosted Image



#143018 Mycology expert ! Identification of fungus by plate culture morphology

Posted by badguy on 09 October 2012 - 12:24 AM in Microbiology

do a slide culture.
it looks like aspergillus to me



#143009 competent cells: CaCl2 autoclave vs filter sterilisation

Posted by badguy on 08 October 2012 - 07:00 PM in Molecular Biology

i did both types of sterilization and both also works good.
but usually i did filter sterilization because it saves timePosted Image



#142171 Lysis method for single human embryonic cell

Posted by badguy on 24 September 2012 - 06:58 PM in Molecular Biology

I never thought of that. lolPosted Image
I'l update you guys after do the PCRPosted Image



#142099 Lysis method for single human embryonic cell

Posted by badguy on 24 September 2012 - 02:01 AM in Molecular Biology

I dont know any DNase inhibitor that would not interfere PCR.
any advice?



#142096 Lysis method for single human embryonic cell

Posted by badguy on 23 September 2012 - 11:33 PM in Molecular Biology

I need help from you guys regarding lysis method for single human embryonic cell.

Other than using Proteinase K and mild detergent (eg. tween-20), is it posible to use boiling lysis method or heat denaturation just like in colony PCR?
I will use this sample to do PCR. Thanks in advancePosted Image




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