Trả lời mọi thắc mắc của bạn

Liệt kê những bài viết mới nhất

Bài viết mớiNgười viếtVào lúc
paltalk code HdAd
Sun Mar 06, 2016 1:28 am
vb ListView HdAd
Sun Mar 06, 2016 1:00 am
Find occurent of character in string nth HdAd
Sat Jan 16, 2016 10:48 pm
Send listbox items to textbox HdAd
Wed Jan 13, 2016 9:55 pm
[VB] Find word in listbox items HdAd
Wed Jan 13, 2016 7:55 pm
code cho hint2 HdAd
Wed Jan 13, 2016 3:22 pm
[vb] Font RTB HdAd
Tue Dec 29, 2015 10:29 am
[vb] selected text RTB HdAd
Tue Dec 29, 2015 9:06 am
[vb] send unicode text HdAd
Tue Dec 29, 2015 9:03 am
[vb] format multi richtextbox HdAd
Tue Dec 29, 2015 9:01 am
Trang web tai cac bang sang che HdAd
Mon Dec 28, 2015 1:28 pm
[VB] Sử dụng resource của chính chương trình HdAd
Sat Dec 26, 2015 8:56 pm
[vb] Phân biệt class và Module HdAd
Thu Dec 24, 2015 2:39 pm
[VB] MY API and Functions declaration HdAd
Mon Dec 21, 2015 1:45 pm
[vb] Khai báo và sử dụng hàm Setforeground HdAd
Mon Dec 21, 2015 1:29 pm
[VB] Sử dụng hàm Sendmessage để gửi tiếng việt, và format richtextbox HdAd
Mon Dec 21, 2015 1:26 pm
[VB] Find menu context handle/ID by SPY++ HdAd
Fri Dec 18, 2015 1:20 pm
[VB] Find menu context handle/ID - Vu Hai Ninh HdAd
Fri Dec 18, 2015 1:00 pm
[VB] Find menu context handle/ID HdAd
Fri Dec 18, 2015 12:54 pm
[VB] Sleep in VB program HdAd
Thu Dec 17, 2015 7:48 pm
[VB] CLose window if found HdAd
Wed Dec 16, 2015 7:07 pm
[VB] Không cho thay đổi kích cỡ form khi chạy HdAd
Wed Dec 16, 2015 3:28 pm
[VB] Không cho thay đổi text box khi chạy HdAd
Wed Dec 16, 2015 3:22 pm
[VB] Tìm cửa sổ và Button bằng FindWindow & FindWindowsEx HdAd
Wed Dec 16, 2015 3:16 pm
[VB] Xóa text mặc định của text box khi click HdAd
Wed Dec 16, 2015 3:12 pm

You are not connected. Please login or register

Chú ý khi làm transformation (vào competent cells)

Go down  Message [Page 1 of 1]



maud63 wrote:I would like to know how you make your bacterial transformation :
- electroporation or chemically competent cells?
- Do you purify your ligation reaction before use (and how?) or do you directly use it? Which maximum volume of ligation reaction for which bacterial volume?
- how many ng of DNA?
- Do you thing it is better to heat inactivate the ligase before transformation?

Thank you for your tips to have the best transformation efficiency.

Trả lời:
What a Face

scolix wrote:If u have an apparatus for electroporation then go for it, as it gives better transformation.

I donot purify my ligation mixture but use 2ul-3ul (out of a total 20ul) of the ligation mixture for transforming chemically competent cells. If its electroporation then 1-1.5ul.

The amount of DNA varies a lot in my liagtion. Normally I use between 20-40ng in my ligation but I have used upto 200ng for ligations.

I donot think it as necessary to inactivate ligase before transformation. As I leave the ligation mixture on the bench so that I could use it the next day if something was went wrong.


Jou wrote:I remember cloning with TOPO vector from Invitrogen using electro-competent cells. Didn't work, because I didnt' purify the ligation and it still contained too much salt from the Invitrogen-supplied ligation buffer.... but usually I don't purify.

Evil or Very Mad

phage434 wrote:Our tests show that chemical transformation with ligation mixes should add less than 5% by volume of ligation mix -- more is inhibitory. We routinely use 1 ul with 20 ul of cells. Heat killing ligase appears to be optional from our experience, but fatal if you are using PEG containing ligation buffer (quick ligation kits). We do not purify our ligation reactions, although we could probably get more transformants by doing so. For electroporation, the main limitation on added ligation mix is the increased conductivity of the cells, which can lead to arcing. Dialysis on floating Millipore membranes over TE or water helps a great deal in this situation.

Concentration of the DNA in a ligation reaction is a major factor in pushing the reaction from intra-molecular to inter-molecular. We usually want low concentrations, since we want to avoid formation of concatamers, which form easily, do not transform well, and are not the desired product. The optimal ratio is a 1:1 molar ratio of insert:vector. We aim for around 20 ng of vector in the 10 ul ligation mix.

Twisted Evil

fred_33 wrote:i use 1/10 of my ligation reaction (for up to 20µl) directly for electroporation.
For heat shock cells, i use the whole ligation directly.

For the TOPO cloning, i use 1 to 2µl of a 1/10° diluted topo reaction for electrocompetent coli.

Finaly, i precipitate ligations with butanol.

View user profile

Back to top  Message [Page 1 of 1]

Similar topics


» translucent cells [HELP]

Permissions in this forum:
You cannot reply to topics in this forum