Team:NRP-UEA-Norwich/Week5

From 2012.igem.org

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==Day 2==
==Day 2==
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. Rachel and Russell calculated the expected length of plasmid and found that the plasmids on the agarose gel were completely different to expected. Decided to carry out a restriction digest of the plasmids to remove the inserts and then run those on a gel to find out if they are as expected. After a nanodrop we found that there was a very small amount of DNA and likely too little to work with; mini-prepped the second samples for each biobrick and nanodropped them.
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Russell calculated the expected lengths of the plasmids and found that they ranged from 3.9kbp (RFP) to 2.3kbp (AraC), however the results in the gel showed the plasmid sizes ranging from 2.5kbp (RFP and AraC2) to 1.5kbp (eCFP and AraC1), a stark difference from what was expected. We decided to carry out two resriction digests of the plasmids; one to linearise them and one to cut out the insert, in order to validate the true size of the plasmid and the size of the desired insert. We then nano-dropped the DNA and found that we had a very small amount of DNA in the solutions. We expected to see DNA levels in the 100s of ng/μl however the results we gained were:
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RFP = 20.0 ng/μl  |  eCFP = 2.9 ng/μl  |  AraC1 = 16.0 ng/μl  |  AraC2 = 2.9 ng/μl
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Clearly a stark difference between the expected result and the actual result. Following this experiment Russell then carried out a DNA mini-prep on the second samples of media containing transformed ''E. coli'', this time using the Bioline Plasmid Isolation kit (Protocol can be found [http://2012.igem.org/Team:NRP-UEA-Norwich/Protocol#Isolating_DNA_.28Bioline.29 Here]). The plasmids that had been isolated from the second DNA mini-prep were then nano-dropped with slightly better results, which were:
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RFP = 28.3 ng/μl  |  eCFP = 46.9 ng/μl  |  AraC1 = 7.4 ng/μl  |  AraC2 = 17.8 ng/μl
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Despite not being in the 100s as desired we decided that we could use this DNA to carry out a future restriction digest and agarose gel run in order to validate it, and then isolate DNA from the ''E. coli'' that was to be transformed by Rachel for the future restriction digest/ligation to produce new biobricks.
 +
 
. Rachel transformed new E. coli with the original biobricks in order to produce a new stock of DNA as we had left gaps between using the bacteria and DNA previously which could account for the very low levels of DNA we had experienced in the nanodrops.
. Rachel transformed new E. coli with the original biobricks in order to produce a new stock of DNA as we had left gaps between using the bacteria and DNA previously which could account for the very low levels of DNA we had experienced in the nanodrops.

Revision as of 11:10, 8 August 2012

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NRP UEA iGEM 2012

 

Welcome to the NRP UEA iGEM 2012 Wiki Lab Book

Please choose the relevant link to access our diary of that week!

Week Zero | Week One | Week Two | Week Three | Week Four | Week Five | Week Six | Week Seven | Week Eight | Week Nine | Week Ten | Week Eleven | Lab Protocols | Experiments

Contents

Week 5

Day 1

Russell and Rachel ran their isolated plasmids on an agarose gel and found that the bands given were not in-keeping with what they expected. They expected the RFP plasmid to be the largest, eCFP the next largest, and then the two AraC plasmids to be smaller yet but the same size as one another; the results showed RFP and AraC2 to be equal to one another and the largest of the four, while eCFP and AraC1 were shown to be equal to one another and the smallest of the four. It is possible that some plasmids were in their super-coiled states and others were not resulting in the abnormal results and it was concluded that further experiments were needed.

Day 2

Russell calculated the expected lengths of the plasmids and found that they ranged from 3.9kbp (RFP) to 2.3kbp (AraC), however the results in the gel showed the plasmid sizes ranging from 2.5kbp (RFP and AraC2) to 1.5kbp (eCFP and AraC1), a stark difference from what was expected. We decided to carry out two resriction digests of the plasmids; one to linearise them and one to cut out the insert, in order to validate the true size of the plasmid and the size of the desired insert. We then nano-dropped the DNA and found that we had a very small amount of DNA in the solutions. We expected to see DNA levels in the 100s of ng/μl however the results we gained were:


RFP = 20.0 ng/μl | eCFP = 2.9 ng/μl | AraC1 = 16.0 ng/μl | AraC2 = 2.9 ng/μl


Clearly a stark difference between the expected result and the actual result. Following this experiment Russell then carried out a DNA mini-prep on the second samples of media containing transformed E. coli, this time using the Bioline Plasmid Isolation kit (Protocol can be found Here). The plasmids that had been isolated from the second DNA mini-prep were then nano-dropped with slightly better results, which were:


RFP = 28.3 ng/μl | eCFP = 46.9 ng/μl | AraC1 = 7.4 ng/μl | AraC2 = 17.8 ng/μl


Despite not being in the 100s as desired we decided that we could use this DNA to carry out a future restriction digest and agarose gel run in order to validate it, and then isolate DNA from the E. coli that was to be transformed by Rachel for the future restriction digest/ligation to produce new biobricks.


. Rachel transformed new E. coli with the original biobricks in order to produce a new stock of DNA as we had left gaps between using the bacteria and DNA previously which could account for the very low levels of DNA we had experienced in the nanodrops.

Day 3

. Rachel checked the transformed bacteria and found that some had grown but others hadn't grown quite as much; left for a few more hours to grow before inoculating.

. Russell carried out a restriction digest of each isolated plasmid hopefully containing the RFP, eCFP or AraC biobricks. He did one digest of each plasmid with just EcoR1 to linerarise the plasmid, and one digest of each plasmid with EcoR1 + Pst1 to cut out the insert. These digested plasmids will then be run on an agarose gel in order to validate the DNA that are in the biobricks due to the unusual results previously; if they are fine then the biobricks transformed into E. coli by Rachel will be used to produce new biobricks and carry out quantitative experiments.

Day 4

Day 5