Ligation Mini Report

Introduction:
In Experiment 2, we will attempt to create a recombinant plasmid containing the
control promoter and Red Fluorescent Protein (RFP) using PCR amplification and
Restriction enzyme digestion to ligate plasmid to RFP and then generate mutant to
measure the changes to RFP expression by measuring RFP fluorescence. The RFP are
inserted into the P1 plasmid through ligation. The P1 plasmid is digested with restriction
and than isolated RFP plasmid is ligated into the plasmid with promoter sequence. In
protocols for experiment 2E through 2I, the main idea is to digest both P1 plasmid and
RFP to isolate the RFP gene with promoter sequence and open P1 plasmids for ligation
and then transform competent cells for ligation analysis. The main goal for sub
experiment 2-2 is to determine the effect of two variables: molar ratio of insert to vector
and removing stuffer from digested plasmid used on ligation efficiency. We hypothesize
that a larger molar ratio of insert to vector will increase the ligation efficiency and
removing the stuffer from the digested plasmid will also increase the ligation efficiency.
Methods
Two reactions will be set up for restriction enzyme digestion of the P1
plasmid and the RFP PCR. For each reaction, 2.5μg of P1 and RFP PCR is added to each
digestion with buffer. Then the restriction enzyme Spe1 and Pst1 is added to the P1
digestion and the Pst1 and the Xba1 enzyme is added to the RFP digestion. Separate the
digested P1 into two part and label one with “P1 with stuffer” and “P1 without Stuffer”.
Then add binding buffer to P1 without buffer. Then the DNA in the sample is eluted into
a microfuge tube using a miniprep column (See Experiment 2B protocol). The three
reactions are placed in a gel with one uncut RFP sample and one uncut P1 plasmid for
electrophoresis in order to determine the resultant RFP digested plasmid did work. After
observation under the UV light, the two digested RFP bands are cut out of the gel and
redissolve for ligation experiment as digested PCR product. The purpose of the gel
extraction is to take the purest digested RFP out of the sample through electrophoresis
since we have uncut RFP as reference. The digested RFP PCR, P1 with stuffer and P1
without stuffer are then used as ligation reactions to determine the effect of
molar ratio of insert to vector and the presence of stuffer. The
ligation reaction for each sample contains 2μl T4 DNA ligase buffer, Vector
DNA, Insert DNA, T4 DNA ligase and nuclease-free water. The controls for the ligations
are one positive control where digested P1 plasmid is ligated with ligase and one negative
control where digested P1 plasmid is ligated without ligase. The ligation reactions are
then put into a vial of cells mixed with cold dilution buffer. For the positive
transformation control, the cells are added with pGEM and for negative control, the cells
are added with water. The ligation reactions are then spread onto separate agar plates
containing ampicillin (to eliminate possible contamination and so that only Amp-R
containing bacteria survives since Amp-R is only present in the P1 plasmid). The plates
are then incubated and the result is analyzed through counting the number of red colonies
and white colonies to determine the percentage red colonies in total colonies and then
from the amount of cells added, the transformation efficiency is determined through an
equation of (number of tranformants/ μg of DNA)* (Final volume at recovery/volume
plated).
Result
 With Stuffer Without Stuffer Controls
P1 cut,
Transformation P1 cut, no
Insert:vector 1:1 Insert:vector 3:1 Insert:vector 1:1 Insert:vector efficiency of (no stuffer
(A) (B) (C) 3:1 (D) positive control ligase) (+ligase)
# # # # #
colonie colonie colonie colonie % colonies/ug of colonie #
s % red s % red s % red s red plasmid DNA s colonies
250.0 204.3 163.2 152.2 69.
Average 9 44.33 5 58.99 1 64.35 3 7 1170352.94 14.62 60.2
244.8 193.5 175.7 144.3 26.
Std 8 25.01 8 24.43 3 22.88 2 65 872022.66 26.56 101.36
Table 1.1 Average number of colonies and percent of red colonies from class data for
each conditions and average transformation efficiency of positive control with average
number of colonies for each positive and negative control without outliers. Outliers are
selected using a p value of 0.05 and according to comments made by each group

treatments Tukey HSD Tukey HSD Tukey HSD

pair Q statistic p-value inferfence

A vs B 2.4401 0.3192919 insignificant

A vs C 3.3321 0.0962567 insignificant

A vs D 4.2235 0.0203502 * p<0.05

B vs C 0.8920 0.8999947 insignificant

B vs D 1.7834 0.5806004 insignificant

C vs D 0.8914 0.8999947 insignificant

Table 1.2 p-value tests to determine the statistical significance of difference between the
four experimental conditions without outliers. A is Insert: vector 1:1 With Stuffer, B is
Insert: vector 3:1 with stuffer, C is Insert: Vector 1:1 without stuffer, D is Insert: Vector
3:1 without stuffer.
Figure 1 Bar plot of average percent Red Colonies with each ligation conditions
with error bars indicating the standard deviation without outliers.

Description:
In table 1, we can see average number of colonies and the percent red colonies for each
reaction condition as well as the standard deviation. These results are determined through
the average of class data without the outliers, which are determined through a critical
value at 0.05 and Group comments on each set of data. From the result we can see that
with an increase in insert: vector ratio, there is an increase in the percent red colonies for
the constant condition of presence of stuffer as with 1:1 ratio and 3:1 ratio. We can also
see that the percent red colonies increase between the presences of stuffer with the factor
of presence of changing as for 1:1 ratio, no stuffer also increase the percent red colonies
and for 3:1, the presence of stuffer increased the percent red colonies. In Table 1.2, it
shows the result of the statistical significance between each treatment through a Tukey
HSD, only the difference between treatment A, which is 1:1 insert to vector ratio with
stuffer is different from treatment D, which is 3:1 insert to vector ratio without stuffer.
This can indicate that only when both conditions, which is no presence of stuffer and a
large insert to vector ratio, are met, can lead to an increase in ligation efficiency. In
Figure 1, it shows the average percent red colonies with error bar as standard deviation
for each condition. As we can see from the plot, there is an increase trend and also we can
see that increasing molar ratio and removing stuffer increases ligation efficiency.

Discussion:
From the results above, we can see that the increase in molar ratio and elimination of the
stuffer will increase the ligation efficiency because for increasing the molar ratio between
the insert and the vector will likely to increase the chance of the insert being successfully
inserted into the vector since there is excessive insert and therefore it is a higher
probability for vectors to close with the insert attached to it. And the elimination of buffer
eliminates the other unwanted material in the sample, which can help increase the
efficiency of ligation through removing the unwanted binding to the vector, causing
resulting expression to be undesired. The purpose of increasing the insert ratio is to
increase the probability of success for ligating insert into the desired vector. Removing
the stuffer will help with ligation since that all undesired DNA will be removed and will
decrease the chance of vector ligating together without the desired vector after digest
since eliminating stuffer leaves the insert to be the only present DNA other than the
vector. For the statistical result, the p value calculated is compared to a critical value of
0.05, showing that once the value falls below 0.05, the difference between the condition
is considered to be statistically significant. In this experiment, only treatment A and D are
considered statistically significant. The effect of outliers in the data set is to lower the
average since most outliers do not have any colonies and can lead to insignificant
statistical difference when conducting p value test. From the result we can conclude that
elimination of stuffer and increase of molar ratio between insert and vector increases the
ligation efficiency, we are unable to conclude that each one contributes to the increase in
efficiency since the p value test is proven to be insignificant but when the two conditions
are both met, it shows that the efficiency is increased and the data difference proves to be
significant since the p value is 0.02, which is below the critical value. To improve ligation
efficiency I propose we can increase the binding affinity between the insert and the
restriction enzyme site so that when the plasmid in cut open, it is more likely to bind to
the insert rather than closing back together by itself.

Supplementary:

Table 2 Raw class data of each experiment condition for ligation. Red highlighted rows
are outliers, determined according to comments by each group indicating failed ligation
and contamination.
Figure 2 Bar plot of average percent Red Colonies with each ligation conditions
with error bars indicating the standard deviation with outliers.