TITLE: PCR Cloning with Blue/White Selection--CHEM 584
AUTHORS: Ken Christensen, Promega, Trevor Wagner

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Protocol for PCR Cloning with Blue/White Selection and Easy Insert Excision using </span><a href="https://www.promega.de/products/pcr/pcr-cloning/pgem-t-easy-vector-systems/?catNum=A1360" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">pGEM®-T Easy Vector Systems.</span></a><span style = ":;"></span></div><div class = "text-block"><span>(For use with </span><a href="https://www.promega.com/products/pcr/pcr-cloning/pgem-t-vector-systems/?catNum=A3600" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">A3600</span></a><span>, </span><a href="https://www.promega.com/products/pcr/pcr-cloning/pgem-t-vector-systems/?catNum=A3610" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">A3610</span></a><span>, </span><a href="https://www.promega.com/products/pcr/pcr-cloning/pgem-t-easy-vector-systems/?catNum=A1360" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">A1360</span></a><span>, or </span><a href="https://www.promega.com/products/pcr/pcr-cloning/pgem-t-easy-vector-systems/?catNum=A1380" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">A1380</span></a><span>.)  </span></div></div>

[STEPS]
?. [Ligation ]
?. [Ligation ]
Set up ligation reactions as follows: Use tubes known to have low DNA-binding capacity. Vortex the 2X Rapid Ligation Buffer vigorously before each use.
2X Rapid Ligation Buffer contains ATP, which degrades during temperature fluctuations. Avoid multiple freeze-thaw cycles and exposure to frequent temperature changes by making single-use aliquots of the buffer.
Longer incubation times will increase the number of transformants. Generally, incubation overnight at 4°C will produce the maximum number of transformants.
Use only the T4 DNA Ligase supplied with this system to perform pGEM®-T and pGEM®-T Easy Vector ligations. Other commercial preparations of T4 DNA ligase may contain exonuclease activities that may remove the terminal deoxythymidines from the vector.
?. [Ligation ]
Mix the reactions by pipetting. Since you will be Blue/White screening, running the control reactions each time is less necessary. The TAs will run negative and positive controls at regular intervals.
?. [Ligation ]
Incubate the reactions for 1 hour at room temperature. Alternatively, if the maximum number of transformants is required, incubate the reactions overnight at 4°C.
[Incubation]
?. [Ligation ]
Analyze an aliquot of the PCR reaction on an agarose gel before use in the ligation reaction to verify that the reaction produced the desired product. The PCR product to be ligated can be gel-purified or purified directly from the PCR amplification using a PCR Clean-Up system.
Cleanup of reactions prior to ligation is recommended to remove primer dimers or other undesired reaction products and to improve ligation efficiency. Exposure of PCR products to shortwave ultraviolet light should be minimized in order to avoid the formation of pyrimidine dimers.
?. [Transformation]
Prepare two LB/ampicillin/IPTG/X-Gal plates for each ligation reaction. To prepare your LB-AMP plates, add  Spread 40 µl of the IPTG stock and 40 µl of the X-gal stock (aliquots are stored in the freezer) on top of the plate with a hockey stick spreader. Equilibrate the plates for 30 minutes at 37″C.
?. [Transformation]
You will get the linearized pGEM-T, Ligase Rapid Reaction Buffer, and DNA ligase from the instructor or TA. Store tube on ice. You will add your PCR product to this mix at a 1:3 ratio of vector (pGEM-T) to insert (your PCR product). You can use the calculator at www.promega.com/biomath. pGEM-T is 3kb and the reaction uses 50 ng of vector. This calculator outputs a 1:1 ratio, so you will need to multiply by 3 to get the mass needed for your PCR product ligation. Then transform into Mix&Go competent cells.
?. [Transformation]
Incubate the plates overnight (16–24 hours) at 37°C.
If 100μl is plated, approximately 100 colonies per plate are routinely seen using competent cells that are 1 x 10^8cfu/μg DNA. Use of ultra-high- efficiency competent cells may result in a higher number of background colonies. Longer incubations or storage of plates at 4°C (after 37°C overnight incubation) may be used to facilitate blue color development. White colonies generally contain inserts; however, inserts may also be present in blue colonies.
Colonies containing β-galactosidase activity may grow poorly relative to cells lacking this activity. After overnight growth, the blue colonies may be smaller than the white colonies, which are approximately one millimeter in diameter.
Blue color will become darker after the plate has been stored overnight at 4°C.
[Incubation]
[Incubation]