Universal T-vector colony PCR identification kit T4 principle carrier

NoboledeUniversal T-vector colony PCR identification kit T4 principle carrier

Universal T-vector colony PCR identification kit

Catalog Number:42015

Composition, storage, and stability of the reagent kit:

Storage: Store at -20 ℃.

The specific product parameters are subject to the parameters in the product manual received

Product Introduction

Colony PCR identification is the most efficient and convenient method for identifying positive clones (including inserted fragments) after T vector cloning and ligation. However, the colony PCR identification kits available on the market use specific T vector primers such as T3, T7, or SP6, which can only be used for identifying positive clones connected to a certain specific T vector. If different T carriers are used, multiple identification kits need to be purchased simultaneously, greatly increasing the cost of use. Our company has developed a universal T vector colony PCR identification kit using universal T vector primers. By purchasing only one kit, it can be used to identify positive clones connected to all common T vectors (including pGEM, pUC, pBluescript series) on the market. In addition, the distance between the primers of colony PCR identification kits using T3, T7, or SP6 as primers and the insertion site is very short. Therefore, when identifying insertions of fragments of about 100bp or smaller, the 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. The distance between the universal T vector primers of this kit and the non inserted fragment should be at least 150bp. Adding the length of the inserted fragment, it is possible to clearly distinguish whether the amplified band is from the inserted fragment or the primer dimer. Avoiding false positives or false negatives greatly improves accuracy.Our company has developed a portable PCR MasterMix premix system that does not require you to add various ingredients one by one. You can directly add single bacterial colonies that need to be screened. After about 1 hour of PCR reaction and electrophoresis detection, the results of recombinant bacterial colony identification can be obtained.

Expected amplification fragment:

Notes:

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

2. When selecting bacterial colonies, a single colony should be chosen, and too many bacterial cells should not be selected to avoid affecting the PCR results.

3. When setting up the PCR program, please determine the extension time based on the size of the inserted fragment. If the inserted fragment size is less than 1 kb, the extension time should be 30-45 seconds. If the fragment is longer, the extension time can be increased proportionally.

Operation steps (using 25)mlFor example, customers can also use 20 systemsmlSystem):

1. Prepare a 1 x Taq MasterMix reaction solution containing primers according to the following system.

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.Place the PCR tube on a 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 was extended to 10 minutes mainly to lyse the colony and release the DNA template.

After the reaction is complete, take 5-10mDirect sample electrophoresis detection.

Note: If the bacteria are difficult to lyse or there are many false negatives due to impurities, try the following method to lyse and release DNA before proceeding.

1. Select bacterial colonies and add 50mL dd water (or 10)mShake in water.

2. 99 degrees Celsius, 10 minutes to inactivate enzymes in the bacterial solution and release DNA.

3. Centrifuge at 12000rpm for 1 minute to remove cell debris.

4. Take the supernatant for PCR, 25mPCR system takes 5mL supernatant (starting at 50)mL dd water), or 1mL supernatant (starting from 10)mUse water as a template.

Universal T-vector colony PCR identification kit T4 principle carrier