Addgene 上的protocol
https://www.addgene.org/protocols/gibson-assembly/
1. Gibson Assembly簡介
構(gòu)建質(zhì)粒是分子克隆技術(shù)中非常重要的一環(huán)。2009年，Daniel Gibson博士和同事發(fā)明了一種簡易的線性DNA連接方法--Gibson Assembly (Nat Methods 2009;6(5):343-5) ，從此造福廣大生物科研汪。
Regardless of fragment length or end compatibility, multiple overlapping DNA fragments can be joined in a single isothermal reaction. With the activities of three different enzymes, the product of a Gibson Assembly is a fully ligated double-stranded DNA molecule. This has proven to be an efficient and effective method for the assembly of plasmids, and molecular biologists now use this method extensively.
不論DNA片段的長度多少、末端結(jié)構(gòu)如何，Gibson Assembly都可以在三個(gè)酶的情況下，讓這些DNA片段在同一反應(yīng)溫度下進(jìn)行完全的雙鏈連接--cool！
2. Why Gibson Cloning? Gibson Assembly的優(yōu)點(diǎn)
- No need for specific restriction sites. Join almost any 2 fragments regardless of sequence.
- No scar between joined fragments.
- Fewer steps. One tube reaction.
- Can combine many DNA fragments at once.
- 不需要特定的限制性內(nèi)切酶位點(diǎn)，幾乎可以連接任意兩種序列
- 連接后無痕跡
- 步驟簡單，一個(gè)管子就能做完
- 可以同時(shí)連接多個(gè)片段
3. Procedure
（1）Design your plasmid and order primers (see figure to the right).
When designing your plasmid, think about what DNA segments you will need to join to create your final plasmid. Adjacent segments should have identical sequences on the ends (sequences A and B in the figures). These identical sequences can be created via PCR with primers that contain a 5′ end that is identical to an adjacent segment and a 3′ end that anneals to the target sequence.
- 想好要使用什么vector，要插入什么片段，可以通過PCR產(chǎn)生互相可識(shí)別的序列。
One strategy is to order primers that are 60 bp long, with 30 bp matching the end of the adjacent fragment and 30 bp annealing to the target sequence.
- 可以設(shè)計(jì)60 bp長的引物，30 bp用于匹配到鄰近片段的末端，另外30 bp用來退火到目的序列。
Avoid strong secondary structures in the homology region. Hairpins in this region can significantly reduce the efficiency of two homologous ends annealing.
- 避免強(qiáng)二級(jí)結(jié)構(gòu)以免降低同源annealing效率
（2）Generate DNA segments by PCR.
（3）Run PCR product on an agarose gel to check for size and yield. If there are significant amounts of undesired product, gel purify DNA segments. Otherwise PCR purification or even the raw PCR mix can work fine in an assembly if you want to save time.
通過凝膠電泳分開PCR產(chǎn)物，通過產(chǎn)物大小辨別目的產(chǎn)物，切膠回收后純化（比較推薦的方法，在PCR產(chǎn)物中若無關(guān)成分不是很多，可以不做純化，也能達(dá)到很好的效果）。
（4）Combine segments in Gibson Assembly Reaction. Note: Yields will be best when the DNA fragments are present in equimolar concentrations. （當(dāng)DNA片段以等摩爾濃度存在時(shí)，產(chǎn)量最好 ）
The Gibson Cloning Master Mix consists of three different enzymes within a single buffer. Each enzyme has a specific and unique function for the reaction:
- T5 Exonuclease - creates single-strand DNA 3’ overhangs by chewing back from the DNA 5’ end. Complementary DNA fragments can subsequently anneal to each other. T5核酸外切酶：在DNA片段的5‘端，產(chǎn)生3’重復(fù)序列。
- Phusion DNA Polymerase - incorporates nucleotides to “fill in” the gaps in the annealed DNA fragments. 高保真DNA聚合酶：合并核苷酸，以“填補(bǔ)”退火后的DNA片段的空白 。
- Taq DNA Ligase - covalently joins the annealed complementary DNA fragments, removing any nicks and creating a contiguous DNA fragment. Taq DNA連接酶：共價(jià)連接退火后的互補(bǔ)DNA片段，去除任何缺口并創(chuàng)建一個(gè)連續(xù)的DNA片段 。
Incubate the mix for 1 hour at 50°C or follow manufacturer's instructions. You can purchase master mix from a company (e.g. NEB or SGI-DNA), or make your own (ex: Miller Lab Protocol).
（5）Transform the DNA into bacteria and screen for the correct plasmid product by Restriction Digest.
將限制性內(nèi)切酶消反應(yīng)后的產(chǎn)物DNA，轉(zhuǎn)入細(xì)菌中篩選目的質(zhì)粒。
（6）Sequence the important regions of your final plasmid, particularly the seams between the assembled parts.
NEB公司protocol
https://www.neb.com/protocols/2012/12/11/gibson-assembly-protocol-e5510
Gibson Assembly? Protocol (E5510)
Protocols.io also provides an interactive version of this protocol where you can discover and share optimizations with the research community.
Optimal Quantities 算好用量哦~
Assembly Protocol
- Set up the following reaction on ice:
- Optimized cloning efficiency is 50–100 ng of vectors with 2–3 fold of excess inserts. Use 5 times more of inserts if size is less than 200 bps. Total volume of unpurified PCR fragments in Gibson Assembly reaction should not exceed 20%. 50-100 ng vector再加上2-3倍的插入片段。如果插入片段小于200 bp，則用5倍。PCR未純化產(chǎn)物的總體積最好不要超過20%。
- Control reagents are provided for 5 experiments. 提供5種對照試劑？
- If greater numbers of fragments are assembled, additional Gibson Assembly Master Mix may be required. 如果要組裝的片段太多，可能會(huì)需要額外的 Gibson Assembly Master Mix
-
Incubate samples in a thermocycler at 50°C for 15 minutes when 2 or 3 fragments are being assembled or 60 minutes when 4-6 fragments are being assembled. Following incubation, store samples on ice or at –20°C for subsequent transformation.
Note: Extended incubation up to 60 minutes may help to improve assembly efficiency in some cases (for further details see FAQ section).
Transform NEB 5-alpha Competent E. coli cells (provided with the kit) with 2 μl of the assembly reaction, following the transformation protocol.
