Universal T-vector colony PCR identification kit T4 principle carrier

Universal T-vector colony PCR identification kit

Universal T-vector colony PCR identification kit T4 principle carrier

Catalog Number:42015

Composition, storage, and stability of the reagent kit:

Storage:-20 ℃ 保存。

Product Introduction

colonyPCRAppraisal isTThe most convenient and efficient method for identifying positive clones (including inserted fragments) after vector cloning and connection. But the bacterial colonies that can be seen on the marketPCRThe identification kit usesT3,T7orSP6Waiting for specificityTVector primers can only be used for a specific type of primerTIdentification of positive clones connected to vectors. If using different methodsTThe carrier requires the simultaneous purchase of multiple identification kits, greatly increasing the cost of use. Our company adopts universalTUniversal development of carrier primersTCarrier colonyPCRIdentification kit, only one type of kit needs to be purchased to be used for all common products on the marketTcarrier(includingpGEM，pUC，pBluescriptseries)Identification of positive clones connected. In addition, adoptingT3,T7orSP6Bacterial colony for primer preparationPCRThe distance between the primer of the identification kit and the insertion site is very short, so in the identification process100bpWhen inserting left, right, or smaller fragments, the primer dimer bands of negative clones and the amplification bands of positive clones are similar in size and cannot be distinguished. Often, the bands of primer dimers are regarded as positive amplification bands, resulting in false positives. On the contrary, it leads to false negatives. This reagent kit is universalTThe distance between the carrier primer and the non inserted fragment should be at least150bpAbove, with the length of the inserted fragment added, it can be clearly distinguished whether it is a band amplified by the inserted fragment or a band amplified by the primer dimer. Avoiding false positives or false negatives greatly improves accuracy.A portable tube developed by our companyPCR MasterMixPre mixing system, you don't need to add various ingredients one by one, you can directly add single bacterial colonies that need to be screened, after1About an hourPCRThe identification of recombinant colonies can be obtained by reaction and electrophoresis detection.

Expected amplification fragment:

Notes:

1. When identifying recombinant colonies, labeling should be done before selecting individual colonies for easy identification before use.

2. When picking up bacterial colonies, single colonies should be selected, and too many bacterial colonies should not be picked to avoid affecting themPCRresult

3. settingPCRWhen programming, please determine the extension time based on the size of the inserted segment. If the size of the inserted segment is1 kbBelow, extend the time30-45Seconds is sufficient, if the segment is longer, the extension time can be increased by this ratio.

Operation steps (to25mlFor example, customers can also adopt the system20mlSystem):

{C}1. Prepare primers according to the following system1 x Taq MasterMixReaction solution.

2. After numbering the colonies on the overnight cultured plates, use a sterilized pipette to pick up a portion of the single colonies and add them to the reaction solution. Blow and mix well to ensure that the colonies are fully dispersed into the reaction solution.

An additional negative control tube without bacterial colonies can be set up to facilitate the analysis of experimental results.

3. Translate into EnglishPCRPlace the tube on the thermal cycler and start the reaction under the following conditions. The recommended conditions are as follows:

94℃10 min

94℃30 sec

55℃30 sec 30-33 cycles

72℃0.5-1 min

72℃5 min

Attention: Please adjust the extension time according to the size of the inserted segment. The pre denaturation time has been extended to10Minutes are mainly used to lyse bacterial colonies and releaseDNAtemplate

After the reaction is complete, take5-10mlDirect sample electrophoresis detection.

Note: If the bacteria are difficult to lyse or there are many false negatives due to impurities, try the following methods to lyse and releaseDNAThen proceed.

1. Select bacterial colonies and add them 50ml ddWater (or10ml ddShake in water.

2. 99Degree,10Minute inactivation of enzymes in bacterial solution and releaseDNA.

3. 12000rpmcentrifugation1Remove cellular debris in minutes.

4. Take the supernatantPCR，25ml PCRSystem selection5mlShangqing (Initial)50ml ddWater), or1mlShangqing (Initial)10ml ddUse water as a template.

Universal T-vector colony PCR identification kit T4 principle carrier