Patrick Kratschmer

6 Questions 10 Answers 0 Followers

Questions related from Patrick Kratschmer

What are good methods/protocols/kits for RNA extraction from Drosophila melanogaster brains for RNA sequencing?

I am planning to extract RNA from Drosophila melanogaster heads, which will be used for RNA sequencing to determine transcriptional changes across specific mutants. Methodologies for RNA...

10 October 2016 9,519 6 View

How to avoid problems using BsiWi restriction digestion?

Hi, I used BsiWi to digest at 55oC for ~4h, but get smears on my agarose gel. It looks like BsiWi didn't properly cut my plasmids. Since heat-inactivation is at 65oC, I was wondering how crucial...

04 April 2016 3,126 3 View

Did anyone else experience low DNA yield using Wizard SV Gel and PCR Clean-Up System on linear 11.7kb DNA?

Hi, I used the above stated kit to purify restriction digestion (BsiWi, 55oC, single-digest, 4h) products (11.7kb for one reaction, ~3.5kb for the other), and got unexpectedly low yield: ~70 % for...

04 April 2016 7,114 6 View

Did anyone experience problems with Acc65I/NotI double-digestion?

Hi, I don't see band saparation on my 1 % agarose gel, after digesting with these two enzymes for ~15h over night @ 37oC, and was wondering whether this combination might have caused issues with...

04 April 2016 8,899 10 View

Could centrifuging at 4°C during DNA purification be the reason for large 260/230 ratio?

Hi, I am using the Wizard SV Gel and PCR Clean-Up System to purify restriction-digested DNA from a 1 % agarose gel. I followed the protocol rigorously, but set the centrifuge at 4°C for all...

04 April 2016 6,946 6 View

Would a 250bp DNA fragment necessarily outcompete a 2.5kb fragment during ligation?

Hi, I double-digested a plasmid to obtain a 2.5kb DNA fragment that I want to insert into a larger plasmid (11.7 kb). However, I failed to see that my double digest also yields a 250bp fragment...

04 April 2016 3,730 10 View