I wanna to construct plasmid using golden gate assembly, I haven,t used it before. I have three insert (each lenght is about 180 bp) and one plasmid backbone (Lenghth 3.5kb). Total four fragements. Now I have a question that how much concentration of Each fragement shoud be mixed for plasmid construction and second question I am also using Dpn1 to get rid from the plasmid which i used as template therefore Now I am confused that Dpn1 will be worked together with same buffer used for Bsa1 or not. third i search online for the protocol but i find different protocol therefor if someone have the best protocol which he/she used before please share with me. Thanks
Hello, Golden Gate cloning technology use Type IIS restriction enzymes are unique from "traditional" restriction enzymes in that they cleave outside of their recognition sequence, creating four base flanking overhangs. Since these overhangs are not part of the recognition sequence, they can be customized to direct assembly of DNA fragments. When designed correctly, the recognition sites do not appear in the final construct, allowing for precise, scarless cloning.
Bette to start with Addgene plasmid.
See likes below for more info good luck.
https://blog.addgene.org/plasmids-101-golden-gate-cloning
https://bio-protocol.org/e2059
Hello, Golden Gate cloning technology use Type IIS restriction enzymes are unique from "traditional" restriction enzymes in that they cleave outside of their recognition sequence, creating four base flanking overhangs. Since these overhangs are not part of the recognition sequence, they can be customized to direct assembly of DNA fragments. When designed correctly, the recognition sites do not appear in the final construct, allowing for precise, scarless cloning.
Bette to start with Addgene plasmid.
See likes below for more info good luck.
https://blog.addgene.org/plasmids-101-golden-gate-cloning
https://bio-protocol.org/e2059
I do not understand what you mean by "having the best protocol". It is a matter of what works. I do not know what protocol you used but here is a manual that i rely on.
Yes, it will work; Dpn I is quite resilient when it comes to reaction conditions. What buffer are you using specifically for the Bsa I digestion?
Thanks every One for your kind reply,
Dear Martin,
I am using the following method
(STEP1)
• PCR reaction mixture 10 μL (about 1 μg of DNA)
•Water, nuclease-free up to 30 μL
•10X Buffer G 3 μL
•Dpn1 and Bsa1 1μL+ 1μL
• Mix gently and spin down for a few seconds.
• Incubate at 37°C for 1 hours.
(STEP2)
•Linear vector DNA 20-100 ng 1 μL
•Insert DNA 1:1 to 5:1 1+1+1 μL
•10X T4 DNA Ligase Buffer 1 μL
•T4 DNA Ligase 1 Weiss U
•Water, nuclease-free (#R0581) to 10 μL
•Total volume 10 μL
(STEP3)
PCR
37 C (1hr)
16 C (5m)
After finishing the STEP1 when I purify the DNA from the gel the concentration is about 30-50 ng/μL or some time below then 30, Then I do the STEP2 i have tried three time but not sucessful now i don,t know that the concentration of DNA is low or some other problems. after transformation I got about 150 colonies. but after colony Pcr most of them have the plasmid which i used as a template, Second; Some of colony give the DNA band on the gel about 200 bp and 400 bp but desired DNA band is 600 bp. Now I don,t know only one fragement is inserted which is about 200 or Two fragment is inserted which is about 400, Is it posssible?
@ Said Nawab,
I do not have extensive experience with GG cloning, so take my advice with a grain of salt. Unless you have considerable non-specific amplification, I would try to get rid of the gel purification step (after all, you are already killing most of the original template via Dpn I), especially if you are irradiating the fragments with UV during purification. Just do a clean up with some silica-based kit.
Also, make sure the ligase and the ligase buffer are OK. Use a plasmid digested with a single enzyme and set up parallel +/- ligase reactions. After transformation, you should get about 100-fold more colonies in the reaction with T4 ligase.
And of course, set up test reactions to see if the Bsa I and Dpn I enzymes are active.
How are you doing the sreening, by using vector primers flanking the insert site?
Hope it helps!
- Badges
- Science topic