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Plasmid Miniprep Experiment

Aims

The aim of this experiment is to extract pSB1C3 and [http://partsregistry.org/Part:pSB1AK3 pSB1AK3] with [http://partsregistry.org/Part:BBa_B0014 BBa_B0014] from E. coli DH5α cells using the Qiagen miniprep kit. The extracted DNA is then analysed using the Nanodrop machine.

Materials and Protocol

Please refer to: Minipreps for Qiagen miniprep protocol, Nanodrop Spectrophotometer for nanodrop protocol and Restriction digests for restriction digestion protocol.

Result

pSB1C3 (No. 1)	pSB1C3 (No. 2)	pSB1C3 (No. 3)	pSB1C3 (No. 4)	lacI (No. 1)	lacI (No. 2)	Double terminator (No. 1)	Double terminator (No. 2)
44.0 µl/ml	19.9 µl/ml	25.0 µl/ml	30.8 µl/ml	10.0 µl/ml	44.2 µl/ml	9.2 µl/ml	39.7 µl/ml

Table 1: Nanodrop spectrophotometer results. The table represents the amount of plasmid present in µl/ml quantity.

Discussion

In our experiment, the concentration of our plasmid DNA range from 19 µg/ml to 44 µg/ml. This value is lower than the expected value of approximately 150 µg/ml for plasmid DNA extraction. The 260/280 nm ratio for all the samples is in the range of 2.0 to 2.4. This indicates that our sample are of low purity.

Conclusion

We are able to obtain plasmid DNA, however the concentartion and the purity of the samples are low. This could be due to the following reasons:

The buffers might have been contaminated
RNAse enzyme might have degraded over time

Solution for the problem

If the buffers of the Qiagen miniprep kit is contaminated, then we will be using the Promega miniprep kit.
If the RNAse enzyme was degraded, then we will be adding 10 µl of fresh RNAse into the P1 buffer.

@@ Line 4: / Line 4: @@
 ==Aims==
-The aim of this experiment is to extract plasmid DNA pSB1C3, pSB1AK3 and plasmid containing ''lacI'' Biobrick from ''E. coli'' DH5α cells with the help of Qiagen miniprep kit and confirming the extraction with the help of nanodrop experiment.
+The aim of this experiment is to extract pSB1C3 and [http://partsregistry.org/Part:pSB1AK3 pSB1AK3] with [http://partsregistry.org/Part:BBa_B0014 BBa_B0014] from ''E. coli'' DH5α cells using the Qiagen miniprep kit. The extracted DNA is then analysed using the Nanodrop machine.
 ==Materials and Protocol==
-Please refer to: [[Team:Newcastle/Minipreps| Minipreps]] for Qiagen miniprep protocol and [[TeamNewcastleNanoDrop Spectrophotometer| Nanodrop Spectrophotometer]] for nanodrop protocol.
+Please refer to: [[Team:Newcastle/Minipreps| Minipreps]] for Qiagen miniprep protocol, [[TeamNewcastleNanoDrop Spectrophotometer| Nanodrop Spectrophotometer]] for nanodrop protocol and [[TeamNewcastleRestriction digests| Restriction digests]] for restriction digestion protocol.
 ==Result==
-* '''Lane 1''': 1kb DNA ladder
-* '''Lane 2''': Extraction of pSB1C3 plasmid (No. 1)
-* '''Lane 3''': Extraction of pSB1C3 plasmid (No. 2)
-* '''Lane 4''': Extraction of pSB1C3 plasmid (No. 3)
-* '''Lane 5''': Extraction of pSB1C3 plasmid (No. 4)
-* '''Lane 6''': Extraction of plasmid containing lacI (No. 1)
-* '''Lane 7''': Extraction of plasmid containing lacI (No. 2)
-* '''Lane 8''': Extraction of pSB1AK3 plasmid containing double terminator (No. 1)
-* '''Lane 9''': Extraction of pSB1AK3 plasmid containing double terminator (No. 2)
 {|border=1
 |-
@@ Line 48: / Line 38: @@
 |39.7 µl/ml
 |}
-'''Table 1''': Nanodrop spectrophotometer experiment result. Table represents the amount of plasmid present in µl/ml quantity.
+'''Table 1''': Nanodrop spectrophotometer results. The table represents the amount of plasmid present in µl/ml quantity.
 ==Discussion==
-We found bands in the lane 2, 3, 4, 5 and 6 showing the presence of plasmid in ''E. coli'' DH5α cells. The ideal concentration of DNA calculated using nanodrop experiment is 150 µg/ml but in the table 1, where all the values have been less than 150 µg/ml which shows that even though there is plasmid present in the cells but it is present in very low amount. One possible explanation for this to happen could be that when the transformed ''E. coli'' DH5α cells were grown overnight for the plasmid extraction protocol, the medium in which they were grown did not contain any antibiotics and because of this the cells did not require plasmid which conferred bacteria with antibiotic resistance and this process is called as plasmid shuffle.
+In our experiment, the concentration of our plasmid DNA range from 19 µg/ml to 44 µg/ml. This value is lower than the expected value of approximately 150 µg/ml for plasmid DNA extraction. The 260/280 nm ratio for all the samples is in the range of 2.0 to 2.4. This indicates that our sample are of low purity.
 ==Conclusion==
-This experiment shows that there is plasmid present in the ''E. coli'' DH5α cells but they are present in a very low amount possibly due to plasmid shuffle which could have occurred during overnight growth in the cultures which did not contain antibiotics against which plasmid provides resistance to the cell.
+We are able to obtain plasmid DNA, however the concentartion and the purity of the samples are low. This could be due to the following reasons:
+# The buffers might have been contaminated
+# RNAse enzyme might have degraded over time
+==Solution for the problem==
+# If the buffers of the Qiagen miniprep kit is contaminated, then we will be using the  Promega miniprep kit.
+# If the RNAse enzyme was degraded, then we will be adding 10 µl of fresh RNAse into the P1 buffer.
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Team:Newcastle/2 August 2010

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