Team:AHUT China/Http : //2017.igem.org/Team : AHUT China/Http : //2017.igem.org/Team : AHUT China/protocols
This project consists of 8 steps:
ONE. error-prone PCR
Put the known template of DNA into the tube of PCR, and then add the raw material for error-prone PCR amplification.
Adding the following ingredients in turn in 30μL system:
Putting the system into the PCR and setting the param:
The process of PCR.
TWO. setting the beginning and end
making three systems in 30μL with the following ingredients.
System 1: Selecting the first-PCR sample.
System 2: Slecting the self-prepared template.
System 3: Slecting the self-prepared template.
PCR primer:
System 1: adding 1μL Rna and Fna respectively
System 2: adding 1μL Prefix and Rna respectively
System 3: adding 1μL Suffix and Fna respectively
Taq deoxyribonucleic acid polymerase （5U/μL 2μL
putting the system into the PCR and setting the parameters
THREE.agarose gel electrophoresis
1.Preparation of buffer solution(TAE): 980ml distilled water(measured it with a graduated cylinder)+20mlTAE(50×TAE was diluted to1×TAE), and poured the diluted water into the electrophoresis tank(TAE could be reused in the electrophoresis tank).
2. Preparation of agarose solution: used weighing scale to get powdered agar of 0.45g and poured 30ml TAE to make a agarose solution of 1.5%, then put it into a microwave oven to cook for 2-3 times (30s/times) until the solid was completely dissolved.
3. Added SuperRed/GelRed nucleic acid dye( Gel Staining): added 3μL of nucleic acid dye into the conical flask which contained 30ml of agarose solution, be careful that nucleic acid dye was poisonous and medical gloves were needed.(The nucleic acid dye was combined with nucleic acids in gels so that they displayed nucleic acid banding under ultraviolet lamp)
4. After the solution was cooled to 60℃, poured it into horizontal plywood and solidified it for half an hour;
5. Application of sample: the following ingredients were added to the three gel pores
6. Electrophoresis: Turn on the power of electrophoresis voltage Us=100V electrical current Is=399mA time Ts=3:30
7. Took pictures then got the results: when the gel of electrophoresis had run to the 2/3 of the gel, then stopped electrophoresis. Took out the gel and took pictures under ultraviolet fluoroscopy.
FOUR. gel extraction of DNA
1.First, took two devoid of nucleic acid and 1.5ml centrifuge tube contaminated by nuclease, and heated the water bath to 75℃
2. Cut the agarose gel shangles that containing target DNA under the ultraviolet lamp, and sucked the liquid on gel surface with paper towel and chopped it up. Then put them into three centrifuge tubes respectively and weighed them on the scale.
3. Added Buffer DE-A of three gels volumes into three centrifuge tube respectively, and heated them at 75℃ when mixed well, mixed them discontinuously(every 2-3min), until the gels were completely dissolved(about 6-8min).
4. Added Buffer DE-B of 0.5 Buffer DE-A volume（250μL）and mixed them well, and DNA fragments of this experiment were less than 400bp, adding isopropyl alcohol of one gel volume（166μL）was needed.(The mixture which had added Buffer DE-B turned into yellow, and it turned into yellow homogeneous solution after fully mixed)
5. Transfer the mixed liquid from the centrifuge tube to the DNA preparation tube(put the preparation tube in 2ml centrifuge tube), and centrifuged for 1 min at 13550r/min speed, then abandoned the filtrate.
6. Put preparation tube back to 2ml centrifuge tube, and added 500μL Buffer W1,centrifuged for 1 min at 13550r/min speed, then abandoned the filtrate.
7. Put preparation tube back to 2ml centrifuge tube, added centrifuge tube, centrifuged for 1 min at 13550r/min speed. Then abandoned the filtrate, and used the same method again to wash it with Buffer W2, and centrifuged for 1 min at 13550r/min speed, then abandoned the filtrate.(Made sure that absolute ethyl alcohol has been added to Buffer W2 concentrate according to the prescribed volume of the instructions on the reagent bottle; used Buffer W2 twice to make sure that salinity had been completely removed to prevent its impact on the follow-up experiments)
8.Put preparation tube back to 2ml centrifuge tube, and centrifuged for 2 min at 13550r/min speed.
9. Put preparation tube back to clean centrifuge tube of 1.5ml, and put it open for 1min (volatilized isopropyl alcohol).30μL deionized water（65℃） was added to the central part of the prepared tube, volatilized for 4min in room temperature, then centrifuged for 2 min at 13550r/min speed to elute DNA.
FIVE "Bridge" Experiment
Experimental considerations
We are planning to gather all the "Site" and "Line" in the PCR tube. Based on the Complementary Pairing Principle, all the basic groups pair together on the "Site" and " Line", and form all kinds of DNA chains(which equals to different pathways). Adding DNA polymerase and dNTP, we put all the single stranded DNA in the tube at a proper temperature.
Selecting two PCR tubes to configure two identical systems with the following ingredients.
Finally, added hi-fi 0.8μl Tap DNA polymerase into all systems respectively.
Putting the system to PCR instrument to set param
SIX DNA repairing
Doing repairing gap reaction in PCR
Adding the following materials into a PCR tube（10μl system)
![](:"http://oyeu0gk8v.bkt.clouddn.com/11.png")
Param setting： 16℃ forever
Put it into biochemical incubator after half an hour(16℃） for reserved.
SEVEN. The collocation of medium
Took a 1000ml beaker, and added 760ml deionized water, then began to dissolve it after adding the following materials
tripton 8g
yeast extract 4g
NaCl 4g
Stirred the liquid until the solid was completely dissolved, and added 5mol/L NaOH（160μl) and adjusted pH to 7.0, then used deionized water in constant volume of 800 ml.
Divided the prepared solution into 4 bottles (every bottle was about 200ml), added 3g agar powder to one of the bottles to make solid medium, and the others were made into liquid nutrient medium. Put the well-prepared medium and culture dish that needed to be sterilized to High-Pressure Steam Sterilization Pot to sterilize.
Plate smearing method
1.Before using Bechtop, turned on UV lamp of Bechtop at least for 30 min to sterilize;
2.Turned on the blower and lamp while using Bechtop, and turned off UV sterilamp and sterilized hands and arms, etc.
3.Opened the lid of the pot, and put the medium in conical flask into sterile culture dish which had been sterilized while it was still hot (agar was not congealed at 50℃) on the Bechtop. Put the triangular flask that contained culture dish next to the fire, and opened the breathable film and let the mouth face to the fire. Put the culture dish with the left hand and covered the flame with the dish.(The method of holding dish: Used ring finger and little finger to hold the bottom, used thumb and middle finger to hold the lid so that it was convenient to open and cover the lid with thumb. Before operation, practiced this action until skillful enough.) Put culture dish about 15mL into it quickly through a slot. Covered the dish and shaked it sightly to make the medium was evenly distributed at the bottom of the culture dish, and then placed it on the desktop. After solidification, made them into plate for inoculation of bacteria.
EIGHT. transformation and screening of target gene
After obtaining the target gene, we need to operate the process of enzymil digestion. According to the design of PCR primers, there are both Restriction Enzyme cutting sites for Pst1 and ECORI-hf at the two ends of the target gene. There are also the same sites in the plasmids. So, the two substances react together for two hours, and then the target gene is introduced into the plasmid. In the same system, the connection between them costs 12 hours. Adding the plasmid into the cells which have all abilities. It also means we should put the plasmids with different BP fragments into different tubes. There are 50 cells which have all abilities being on this process for 20 minutes, and then put them into the water in 42 degree Celsius for 40 seconds. Finally, we should coater cells on the soild medium.
1.Choosing E-coil, Chl-medium without target gene and smearing them evenly on the medium.
2.Wrapping E-coil without target gene in the Chl-medium evenly.
3.Wrapping E-coil with digestion of plasmid evenly in the Chl-medium.
4.Smearing E-coil with target gene evenly in the Chl-medium.
After smearing, culture for 16 hours.
After chlturing, observing the growth of E-coil, selecting the liquid medium which we need, and then putting it into the rocking pot to rock for 16 hours.
