Team:University College London/LabBook/Week12


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'''Conclusion:''' We will revise the protocol to see if we can detect any bands.
'''Conclusion:''' We will revise the protocol to see if we can detect any bands.
== 12.3 ==
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Revision as of 00:32, 27 September 2012



Monday 27.8.12

Aim: To find the right concentration of W3110 E. coli cells in LB that would be later used to streak out on the agar plates to obtain an optimal amount of colonies.

Methods: 1. The colony was inoculated the night before in 10ml of LB plus 10 ul CMP 2. The day after 5 dilutions were made from the sample 3. First solution - 1ml of the original solution & 4ml of the LB Second – 1ml of the first solution & 4ml of the LB Third – 1 ml of the second solution & 4ml of the LB Fourth – 1 ml of the third solution & 4ml of the LB Fifth – 1ml of the fourth solution & 4ml of the LB

Tuesday 28/08

Aim: (on Monday grew WNu o/n in prep for miniprep. This miniprep will be used as a template for pcr of a 2nd nuclease.

Not the synthesised staph aureus nuclease (Plan A). The Wnu Nuclease is considered our plan B.

Methods: (10ul cells, 10ul, amp, 10ml (LB). Wnu cells were obtained from a glycerol stock.

Optimal number of colonies was achieved using the fourth solution/dilution (24 colonies). Using the dilution number 4 we plated out 12 plates.


Aim - Amplify irrE from Deinococcus radiodurans.


PCR Protocol

Step 1 - Setting up PCR tubes: Thaw reagents and add to PCR tubes in the proportions described in the table below

PCR Components Volume (ul)
5x Reaction Buffer 10
25mM MgCl2 4
10mM dNTPs 1
10uM Forward primer 5
10uM Reverse primer 5
DNA Polymerase 0.25
Nuclease Free Water 24.25
Template DNA 0.5
Total Volume 0.5

Step 2 - PCR program: Add PCR tubes to a thermocycler and run under the following conditions.

PCR conditions Temp (oC) Time (s)
Initial Denaturation (1 cycle) 95 30
Denaturation/Annealing/Extension (30 cycles) 95/55/72 10/25/120
Final Extension (1 cycle) 72 600
Hold 4

Step 1 - Setting up PCR tubes: The table below gives the identity of the primers used for each reaction.

DNA Template Function Module Primer Pair Primer Primer Sequence
Deinococcus radioduran colony irrESalt Tolerance STF1/ST2RSTF1 atggggccaaaagctaaagctgaagcc
ST2R tcactgtgcagcgtcctgcg
STF3/ST4R STF3 gtttcttcgaattcgcggccgcttctagagatggggccaaaagctaaagctgaagcc
ST4R gtttcttcctgcagcggccgctactagtatcactgtgcagcgtcctgcg
No Template (Negative Control) N/A Salt Tolerance STF1/STF2STF1 atggggccaaaagctaaagctgaagcc
ST2R tcactgtgcagcgtcctgcg


Conclusion: We will revise the protocol to see if we can detect any bands.