Universal cloning kit By Lablead biotech,

详情

LabpO TOPO-TA/Blunt Universal cloning kit CAT：PT***1 $*0 for *0T;$**0 for **0T(negotiable)

Storage：**0℃，Valid for at least *2 months.ce pack transport, **2 days in the outdoor room temperature does not affect the quality.

composition	*0 rxns	*0rxns
TOPO-TA/Blunt Vector(*0ng/ul)	*0ul	**0ul
**0bp Control（0ng/ul）	5ul	5ul
*0 x Enhancer	*0ul	*0ul

Product description： This product is different from the traditional T4 ligase principle, which takes advantage of the principle that Topoisomerase can join DNA fragments in an instant (seconds - minutes) and efficiently (close to **0%) using the company's original process.
1. Any PCR product (compatible with A-end/flat-end) can be connected in an instant (seconds to minutes).
2. The size of the special new vector plasmid is only less than 2kb, giving full play to the advantages that the smaller the TOPO vector is, the larger the fragment can be accommodated, and the efficiency of large fragment liagtion is maximized; The size of the connected plasmid was more than 2kb smaller than that of the traditional vector. The smaller the plasmid, the higher the conversion efficiency, and the number of transformers after the liagtion of various fragments was greatly improved.
3. The recovery time of ampicillin resistant carrier is only *0min, which is 6 times shorter than that of Kana-resistant carrier for 1 hour.
4. The fastest conversion can be completed within 5min at room temperature without ice bath and heat shock; No need for *-hour recovery, only *7℃*0 min recovery can be coated. It takes as little as ****0min from the liagtion to the coating plate.
5. Zero background principle of suicide gene, no self-linking false positive, no cumbersome blue and white spot screening and colony PCR screening. Most of the time a random clone is inserted (close to **0%).
6. The ability to connect long fragments is far superior to the traditional TA/Blunt cloning vector, which can connect fragments up to *0kb (for example, ligation 5kb fragments may reach *0 colonies, and at least 8 are inserted), and is a new generation of the world's leading TOPO TA/Blunt cloning vector with simple, fast and zero background screening.
Notice：Only M*3F/M*3R universal primers can be used for sequencing (see the diagram below), but M*3(**7)/M*3(**8) universal primers cannot be used for sequencing. Colony PCR uses the same primers as sequencing.

Protocol：
1.Preparation of the liagtion reaction：
PCR primers can be used with normally designed primers without any changes (phosphorylation primers cannot be used). Any PCR product (compatible A-terminal/flat-terminal) can be directly connected. PCR products are generally recommended to be recycled and purified as a glue, which can avoid possible subsequent problems. If the PCR product has only target bands, non-specific bands and primer dimers, the direct linking reaction can also be attempted. If the PCR product is a plasmid template, it is best to purify it, because the template plasmid may also grow colonies (but not the target vector that you want to build).
2.ligation
1)Room temperature (*5℃**7℃) set up *0ul liagtion system (0.2ml PCR tube is recommended, PCR instrument temperature control)：

Purified PCR product or1ul ***0bp control	0.**5ul
TOPO-TA/Blunt Vector	2ul
*0 x Enhancer	1ul
ddH2O	Xul
total volume	*0ul

After adding the reagent, gently blow and mix it with a pipette or gently mix the bottom of the tube, and then centrifuge at a low speed to collect all the liquid at the bottom of the centrifugal tube. Note that this step cannot be performed on ice, but only at room temperature (*5℃**7℃).
Notice：If the 5ul system is used, each component can be halved according to the proportion, and the number of uses can be doubled.
Recommended dosage for different size inserts (too much leads to fewer converters) :

Insert fragment size（bp）	Recommended Dosage (ng)
*******0	****0
********0	****0
********0	*****0

2) Room temperature (*5℃**7℃) for 5min. The carrier is recommended to complete the liagtion at room temperature (*5℃**7℃) for 5 minutes. Long segments or difficult segments can extend the liagtion time to ****5min, and the temperature can be selected at *7℃, which can significantly increase the number of converters.
3) Place the product on ice for later use. Immediately follow the standard conversion procedure or the rapid conversion procedure.
3.Rapid transforming：
1) The receptive cells are removed from **0℃, quickly inserted into the ice bath, thawed and melted (about **3min).
2) Immediately add 5ul of the ligation solution (up to all of it can be added, as long as the volume does not exceed 1/*0 of the volume of the receptive cells),
gently mix it with the hand at the bottom of thecentrifugation tube (avoid sucking with a gun), and place it in an ice bath for 5min.
3) *2℃ water bath heat shock for *0 seconds, quickly return to the ice bath to stand for **3min, the process do not shake the centrifuge tube.
Note: This step is preferred for *2℃ water bath for *0 seconds heat shock. However, according to experience, most of the commercial TOP*0 and DH5a receptive cells can also be placed at room temperature (˃*2℃) in this step. The time need not be very accurate, and the centrifuge tube can be placed for about **8min in summer or when the room temperature is higher. If the room temperature is low, the time can be extended to about ***5min. Experienced customers can try the heat shock free conversion according to the specific situation.
4）Add ******0ul LB or SOC medium (without antibiotics) and oscillate for *0min at *7℃ at **0 rpm.
According to experience, it is generally possible to directly add the medium (if the temperature is low when taken out of the refrigerator, it should be placed in a *7℃ temperature box and returned to *2℃**7℃) to the 1.5 ml centrifuge tube of receptive cells, cover the centrifuge tube cover, and fix it horizontally in the oscillating incubator for oscillating culture and resuscitation, without transferring it to tube culture and resuscitation. In general, when commercial receptive cells do not exceed 2kb of inserted fragments, sufficient inverters can be obtained by resuscitation ****5 min after heat shock. If the efficiency of self-made receptive cells is low, or if there are few inverters and long inserted fragments, the resuscitation time can be increased to ****0min to obtain more inverters.
5）Take ******0ul bacterial solution coating plate (culture plate contains ampicillin **0ug/ml), and culture overnight. (If few transformeders are expected, in order to obtain more clones, centrifuge at ***0rpm for 1min, absorb part of the supernatant, retain ******0ul, lightly suspend bacteria, and take all bacterial solution coating plate)

4.Screening and identification of transformants：
This product adopts the principle of zero background of suicide gene, no insertion self-connected bacteria will commit suicide and cannot grow, so there is almost no false positive, under normal circumstances, what you see is what you get, as long as the growth of the colony is normal (not contaminated bacteria, the number of transformers is not too small), basically contains insertion. Therefore, when the inserted fragment does not exceed **3kb, **2 bacteria can be directly selected for sequencing without colony PCR identification.
1）In general, the positive rate of TOPO carrier in our company is very high, so the colony growth is normal and the number is not too small, it is recommended to skip the colony PCR identification and direct sequencing. Note that sequencing primers cannot be sequenced using M*3(**7)/M*3(**8) universal primers.
2）The colony PCR results of TOPO vector were prone to false negative results. Therefore, in the case of colony PCR identification, if the colony PCR result is positive, the result can generally be trusted. If the result is negative, or if the amplification size does not match the expected size, it is generally not believed, and the possibility of false negative colony PCR results should be considered. Further extraction of plasmid electrophoresis run size or enzyme digestion identification is required to confirm.
3）In the case of large insert fragments, the inserted plasmid can be directly identified by running electrophoresis to see the size of the plasmid. The insert fragments can also be released by EcoR I/EcorV single enzyme digestion or cut with other appropriate enzymes, and the fragment size can be checked by agarose gel electrophoresis to determine whether the target fragment is contained.

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產品組 :	Molecular Biology