Team:HKU-Hong Kong/Lab Diaries

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Revision as of 15:54, 4 October 2011

Lab Diaries

Week 1

Transformation of reporter DNA (pEGFP-loxp-km-loxp) into DH10B (non-virulent strain E. coli) with antibiotic resistance (Chloramphenicol – Cm)

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 <OL>
 <LI>Transformation of reporter DNA (pEGFP-loxp-km-loxp) into DH10B (non-virulent strain E. coli) with antibiotic resistance (Chloramphenicol – Cm)</LI>
- <OL>
- <LI>tested the efficiency of transformation at different concentration
- <LI>3ug/uL; 30ug/uL; 300ug/uL
- <LI>spread plates (100uL) and incubated the plates at 37C overnight
- <LI>it was found that 3ug/uL of DNA was already enough to have high transformation efficiency of our DH10B competent cells. However, the colonies were very small. This might be because the bacteria grow very slowly.
- <LI>inoculated 5 colonies from the plate which had been spread with 3ug/uL reporter DNA transformed DH10B separately into 3mL LB broth with 3uL Amp and incubated at 37C for several hours to see if it could really grow in growth medium
- <LI>failed results because DH10B was Cm resistant but not Amp resistant</LI>
- <LI>inoculated 5 colonies from the plate which had been spread with 3ug/uL reporter DNA transformed DH10B separately again. But this time into 3mL LB broth with 3uL Cm and incubated at 37C overnight
- <LI>failed results, no turbidity in all the tubes. Perhaps it was because the bacteria grow too slowly. So we put it on the rotary for longer time. There was still no results, no turbidity. It might be because the colonies are so small that when we picked the colonies for inoculation, the bacterial cells could not be picked up by the tips and still stuck to the agar plate; or, the bacterial cells get stuck to the tips without really getting into the LB broth.
- <LI>Inoculated again and plasmid extraction (using mini-prep) was carried out.</LI>
-  <OL>
-  <LI>electrophoresis (confirmation of successful extraction)
-  <LI>enzyme digestion using NdeI and EcoRV separately for further confirmation of plasmid size</LI>
-<table border="1">
-<tr>
-<td>DNA</td>
-<td>2uL</td>
-</tr>
-<tr>
-<td>NE buffer 4</td>
-<td>2uL</td>
-</tr>
-<tr>
-<td>NdeI</td>
-<td>0.5uL</td>
-</tr>
-<tr>
-<td>ddwater</td>
-<td>15.5uL</td>
-</tr>
-<td>Total</td>
-<td>20uL</td>
-</tr>
-<tr>
-<td>DNA</td>
-<td>4uL</td>
-</tr>
-<tr>
-<td>NE buffer 3</td>
-<td>2uL</td>
-</tr>
-<td>BSA</td>
-<td>0.2uL</td>
-</tr>
-<tr>
-<td>EcoRV</td>
-<td>1uL</td>
-</tr>
-<tr>
-<td>ddwater</td>
-<td>12.8uL</td>
-</tr>
-<tr>
-<td>Total</td>
-<td>20uL</td>
-</tr>
-</table>
-<LI>electrophoresis
-<LI>incorrect band sizes for EcoRV digestion