Team:BYU Provo/Notebook/Phage Purification/Fallexp/Period3/Dailylog

Phage Purification

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July-August

September-October

- 10.21 T4 PCR

AB DL AC

10/23/13

-Ran a gel on the PCR we did last time.

-Gel

100 mL TAE 1x

1 g regular agarose

2 drops ethidium bromide

Combine and melt TAE and agarose

Cool and add 2 drops ethidium bromide

Allow gel to form with 14 well mold (20 minutes in the fridge)

-Preparing machine

Fill machine with TAE buffer until it covers gel

2 microL DNA ladder in first well

2 microL stain and 5 microL sample, mix and put in well

Run at 130 V for 60 minutes

We place the samples in the order: DNA ladder, 297/298 W M, 301/302 W M, 311/312 W M, 313/314 W M W=wildtype M=mutant

We got bands for both the 311/312 and 313/314 wild type and mutant. We need to do PCR cleanup and start with the cloning procedure. 297/298 and 301/302 didn't show any bands for either the wild type or the mutant. We suspect this may be to boiling them for too long (12 min) at the start. We are going to try boiling them for only 5 minutes and see if we can get bands next time.

The PCR products were stored in the Cholera QS II box in the freezer.

AB DL AC

10/25/13

-Today we ran PCR again on T4 wild type and mutant phage to determine the sequence differences in the capsid proteins.

- 10.25 T4 PCR

AB DL AC

10/28/13

- We ran another PCR attempting to get bands for sequencing both the major and minor capsid proteins. We used our normal protocol except did not boil the phage this time. Skip also used the protocol he normally uses to see if reagents are the problem with us not getting a band.

- We sent in the major and minor capsid protein genes of the wild type and mutant for sequencing.

AB DL AC

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