Ligation & Transformation lab PDF

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summer molecular and biotechniques (keep in mind notebook results might differ but my explanation can be helpful to explain your own concepts)....

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120:452 Molecular Biotechniques Ligation and Transformation June 18, 2020 Introduction Ligation is a process of joining two DNA fragments by the formation of a phosphodiester bond, while transformation is the process of transferring the DNA plasmid into bacteria. In restriction digest, we isolated the PCR product actin and the GFP plasmid with restriction enzymes, EcoRI and Sac II to obtain hybridized compatible sticky ends. The purpose of “Ligation and Transformation” is to join together the digested PCR product (actin) and EGFP plasmid DNA fragments through the formation of a phosphodiester bond and create a continuous coding sequence of green fluorescent protein (GFP) and actin. A 3:1 ratio of PCR product actin to EGFP plasmid was used to increase the likelihood of getting the insert into the plasmid. Since the plasmid isn’t able to replicate itself, it will be inserted into bacteria for later sequencing by selecting bacteria that have taken up the plasmid by plating them into LB Kanamycin plates. Procedure Before beginning with our ligation experiment, we must thaw all reagents on ice to avoid from pipetting anything that will mess up the reaction. We will also need to set up the ligation reactions on ice to guarantee maximal enzymatic activity and survival. To set up the ligation reaction, we will need to calculate the volumes of the vector (pEGFP) 3-30 fmol and insert (actin) 9-90 fmol. To do this, we will need to obtain the concentration of the digested DNA by comparing the brightness of our bands in CCg and CCp against the Low Mass Ladder (lml) from our restriction digest gel electrophoresis results. Since both CCg and CCp have the same brightness as the 400 bp band in the lml, to calculate the concentration, we divided 40 ng by 3 microliter (uL) to get 13.3 ng/uL for both the plasmid and actin concentration. Then, to calculate the volume needed for the reaction, we divide the number of DNA pieces by our DNA concentration. Using the masses in the 30:80 ratio fmol column, our calculated volumes were 3.5 uL or the EGFP plasmid and 2.6 uL for the PCR product actin. After obtaining the proper needed volumes from our vector and insert, we can set up and complete the ligation reaction. To do this follow these steps: add 2 uL of 10x Ligase reaction buffer, 3.5 uL of vector (pEGFP), 2.6 uL of insert (actin), 10.9 uL of water, and 1 uL of Ligase. The ligation reaction should have a total volume of 20 uL. This reaction is for the experimental reaction, which contains both the plasmid (vector) and the actin (insert) in the ligation reaction. Following the experimental, we set-up three different controls for the ligation reaction. This is because the controls will tell us if our plasmid have any contamination or if it hasn’t been fully digested by the restriction enzymes. First, for the vector (pEGFP) alone use 3.5 uL of plasmid, and replace the 2.6 uL of actin with water. Second, for the insert (actin) alone use 2.6 uL of actin, and replace the 3.5 uL of plasmid with water. Third, for the 1xcut vector alone, use 3 uL of plasmid show. For an undigested vector, nothing should be added. The undigested vector is used for our transformation reaction to make sure that our cells are competent. After setting up the ligation reaction and its controls, we incubated them at room temperature for 30 minutes. Then we obtained competent cells and keep them on ice. Now, we can start our transformation reaction to transfer the plasmid DNA into bacteria. Before starting the transformation process, gently add 5 uL ligation reaction to 50 uL of competent cells Here, we will be working on 3 different transformation reaction: GFP+actin, actin alone, and GFP alone. After setting these reactions on separated tubes, incubate them on ice

Ligation and Transformation June 18, 2020 for 30 minutes. After 30 minutes, proceed to heat shock the cells at 42 degree Celsius for 20 seconds. Do not pass 20 since heat shock pass 20 seconds can destroy and damage the DNA. After heat shock, place on ice for 2 minutes. Then add 950 uL of recovery media to each tube, which will help the cells to fix their cell walls and start cell division to copy our plasmid. To help the cells with recovery, we incubated the cells at 225 rpm at 37 degree Celsius for 60 minutes. After this step, we plate the cells onto LB Kan plates and select for cells with KAN antibiotic resistance to avoid obtaining satellite plasmid. For the experimental reaction, we plate the cells, 50 uL on one LB Kan plate, and 200 uL on the other LB Kan plate. For the control, we plated 200 uL of cells on LB Kan plate. These reactions were incubated overnight at 37 degree Celsius. These plates were removed from the incubator 16 hours later and the number of colonies on each plate were counted. Results

CC

1x digested pEGFP pEGFP

Digested Actin + Digested pEGFP 50 uL plate: 7 colonies 200 uL plate: 44 No colonies --

Digested actin alone No colonies

--

Digested pEGFP alone No colonies

100+ colonies Lawn of cells

Figure 1: The table above shows the number of colonies on the experimental transformation reaction and the control plates. the results above show us if the plasmid contaminated the sample or if the plasmid was not fully digested by the restriction enzymes. The cells were plated on LB Kan plate. Colonies were selected from plasmid with the KAN antibiotic resistance. It can be observed that the digested actin and plasmid in the 50 uL plate contained 7 colonies, while in the 200 uL plate, it contained no colonies. For the controls, the digested actin and the pEGFP alone, no colonies were found. The 1x digested pEGFP had 100+ colonies and our pEGFP had lawn of cells. ng DNA = fmol DNA ends x (1 ng/3000 fmol) x (size of DNA in kb/1 kb) 40 ng / 3 uL = 13.3 ng DNA concentration 35ng/(13.3ng/ uL) = 2.6 uL actin 47ng/(13.3ng/ uL) = 3.5 uL plasmid Figure 2: results to the above shows the calculation of our needed volumes for the reaction, using the masses in the 30:90 fmol for our number of DNA pieces and dividing by the concentration of our digested actin and plasmid.

Ligation and Transformation June 18, 2020 Discussion The results show us whether or not the plasmid was digested by the enzymes and if our plasmid was taken up by the bacteria for later sequencing. We expected to have colonies with antibiotic resistance to Kanamycin, which matched our results. We obtained 7 colonies in 50 uL plates with LB Kan antibiotic. To further analyze our results, we used controls. In the digested actin alone, we obtained no colonies because our gel samples from the restriction digest results, CCg and CCp contained no additional template in the sample. This meant no plasmid was still around after purification and digest, and no colonies would be plated out. Furthermore, in the digested pEGFP alone control, no colonies were obtained because the plasmid was cut by both restriction enzymes. This means that the plasmid contains two different overhang ends in their base sequence because both EcoRI and Sac II cut the plasmid. This will not allow the bases to pair and hybridize, and therefore no ligation will be able to occur. However, by using a 1x digested pEGFP control, we obtained 100+ colonies which showed us that ligase worked, at the AATT- overhangs for the restriction enzyme, EcoRI. This means our ligase was able to bind both the PCR product actin and EGFP plasmid, and hence we obtained colonies of both digested actin and pEGFP. Furthermore, we know that our selected bacteria were able to take up the plasmid and its DNA because our cells were competent and were able to take DNA and give us a plasmid since some grow of colonies were experience on the plates. This means that after transformation, we will be able to culture single colonies in LB media with antibiotic to later selectively pressure and have the bacteria amplify our new GFP-actin sequence for us....