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There were only 6 colonies. So the experiment must not really have the promoter. There would be more colonies.

Overall we didn't successfully clone our vector and insert. Since we had some colonies for our pBR32 only(control). So therefore we can assume we successfully could clone part of our vector. The problem we probably didn't clone the insert completely right. Something went wrong with the ligation process. So, to fix this we would begin again with a focus on the ligation process. So, to fix this we would begin again with a focus on the ligation. Since we may need to remove or play with the insert in a different way. Example: PCR purification, gel and heat inhibition. We were given 20 uL of pBR9 Clade C

# data[].attributes.body
Overall we didn't successfully clone our vector and insert. Since we had some colonies for our pBR32 only (control) so, therefore we can assume we successfully could clone part of our vector. The problem we probably didn;t clone the insert completely right. Something went wrong with the ligation process. So, to fix this we would begin again with a focus on the ligation. Since we may need to remove or play with the insert in a different way. Example: PCR purification, gel and heat inhibition.

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Transformation protocol (Based on NEB C2988 for DH5Alpha Subcloning Efficieny Competant cells) procedure

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DNA Ligation
We had worked on our insert and vector calculations. Based off of our PCR purification results and Gel-electrophoresis results, we had estimated about 40ng of PCR, about 33ng of Gel-electrophoresis. Also since we are using a 3:1 ratio we have to use the NEB calculator to determine the best ligation insert DNA length 1 kb, vector DNA 4.3 kb and 50 ng is what we are going to keep the vector DNA mass. The 3:1 ratio result is 34.88ng, for our insert.
Calculations for insert:
40ng/5ul = 8ng/ul
34.88ng/2 = 17.44ng
17.44ng/8ng/ul = 2.2ul
Calculations for vector:
33ng/4ng = 8.25ng
8.25ng/5ul = 1.6 ul

*Since our solution had such a faint result for our gel. We won't be using a control tube for our experiment. Instead we'll just do one experiment tube.

Experimental Tube:
2.2 ul - insert
30.3 ul - vector
4.0 ul - 10x T4 DNA Ligase Buffer
2.5 ul - Nuclease free water
1 ul - T4 DNA Ligase

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For our PCR purification our agarose gel was not melted enough. Since there were random white dots found in the background.

For our gel-electrophoresis results. We didn't have clear results since some DNA was missing. Since we didn't have a real successful result we will be practicing it in lab again.

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Qiaquick purification column
Calculations performed:
Step 1- Add 5 volumes 50uL * 5mL= 250uL
Step 4- 10ml of PE buffer
50uL * 10ml= 500uL

Steps updated:
1- 50uL * 5mL=250uL
2- Is the same except we follow the triangle steps throughout the procedure, not the circle.
3- We followed the same steps here also.
4- 50uL *10mL= 500uL, follow the rest of the step.
5- Followed the same step.
6- The clean tube was 1.7 ml microcentrifuge tube
7- The beginning of the step followed was the same. We though inserted 6uL cutsmart (XbaI & NsiI)
1uL of N
1uL of X
2uL of H2O
50uL PCR rxn
Overall: 60uL of digest

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Digests and Gel Electrophoresis: procedure

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-For future reference look at the fwd and rev primers, tail ends for primer dimers. Try to avoid creating primer dimers, which are the faint lines below 0.5 kb. Our lane was the 4B we can see that we had a result present. We accessed that the band is about 1 kb.

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.8 ug
1 ul fwd primer
1 ul rev primer
2.1 ul (1:25 dilution gene) = 1 ng of template
25 ul master mix
21 ul H2O