Glasgow/Wetlab/Week8

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Latest revision as of 17:10, 22 October 2007

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Week 8

Monday 20th August 2007

1. Christine did more minipreps for colonies suspected of containing the 7 gene operon phzABCDEFG. When these minipreps were run on a gel there was no evidence of an insert.
2. Maija continued working with phzS in both vectors 4/6B and 3/20G. The minipreps 7.-16. from 17th Aug had been corrected with one of the two PstI sites. With the colonies 7., 8., 9. and 16. another round of site-directed mutagenesis PCR was performed with phzS_SDM_PstI_2 primers. With the colonies 10.-15. mutagenesis was performed with phzS_SDM_PstI_1 primers. After the PCR Maija did a DpnI digestion to get rid of the parental template strand and then transformed TOP10 cells.

Tuesday 21st August 2007

1. Christine cloned phzA→phzG into TOPO vectors using different ratios of PCR product:TOPO vector (see Protocol 12 ). Ratios were 0.5ul:1ul, 1ul:1ul, and 2ul:1ul. Best results were seen at 1ul:1ul.
2. TOPO vectors with different ratios of PCR product:TOPO vector were tranformed (see Protocol 13 (with 30 sec heat shock at 42°C and 250ul of 37°C LB instead of SOC medium))and grown overnight on kanamycin plates at 37°C, each reaction divided into 100ul and ~200ul per plate.
3. Maija did 5 ml LB kanamycin/carbennicillin overnight cultures from 17 colonies from the second round of site-directed mutagenesis for phzS in 4/6B and 3/20G.
4. Maija digested the construction vectors 3/20G and 4/6B, and the 7 gene operon with SpeI and XbaI (Protocol 7) with the intention of then ligating the 7 gene operon into the construction vectors. The 7 gene operon DNA (taken from sample 2 of the PCR which was gel extracted 13/08/07) did not appear on the gel.

   Label	Description	Enzymes	Expected size	Result
   3/20G	Construction vector	SpeI, XbaI	2071bp	2071bp
   4/6B	Construction vector	SpeI, XbaI	2928bp	2928bp
   phzA→phzG	7 gene operon	SpeI, XbaI	6263bp	No result

5. Christine gel extracted 4/6B and 3/20G (see Protocol 11) using 30ul ddH2O at 60°C to elute the DNA.
6. To check presence of the 7 gene operon in the samples of PCR product (gel extracted 13/08/07), Christine restriction digested the sample with EcoRI (see Protocol 7). Expected sizes were 928bp, 2622bp and 2713bp. No DNA was present in the gel. It is now thought that the gel extraction has not worked.
7. Overnights of TOP10 cells prepared for *cells.

Wednesday 22nd August 2007

1. Christine did PCR for phzA→phzG using KOD XL and KOD XL programme (see Protocol 9) with annealing temperature of 55ºC and extension time of 6 mins. Resulted in extremely faint band approximately 7kb in size.
2. Christine also did PCR for phzA→phzD and phzD→phzG using KOD and KOD programme (see Protocol 9) with annealing temperature of 55ºC and extension time of 2 mins. Each gave a clear band approximately 3.5kb in size.
3. Maija miniprepped the 17 cultures of mutated phzS constructs.

Thursday 23rd August 2007

1. GoTaq was added to each of the PCR products of phzA→phzG, phzA→phzD and phzD→phzG from 22/08/07. They were then extended at 72ºC for 10 minutes. Once run on a 1% gel for 30 mins at 100v they were gel extracted (see Protocol 11), cloned into TOPO vectors(see Protocol 12), transformed into TOP10 cells(see Protocol 13)and grown overnight at 37°C on kanamycin plates, each reaction divided into 100ul and ~200ul per plate.
2. Site-directed mutagenesis phzS plasmids were run on a gel. See the first picture here. Minipreps 5 and 9 seem wrong in size.
3. Maija did a PstI restriction digest (see Protocol 7) to show that the site-directed mutagenesis has worked. The picture of the gel is the second one here and it shows that the minipreps labelled as 2, 3, 4, 7 and 9 are right in size and they do not contain any PstI sites. These plasmids were sent for sequencing.
4. Maija did colony PCR from Lynsey's Pr+XylR in TOPO vector transformations from 12 colonies with "Pr_Prefix" and "XylR_Suffix" primers (see Protocol 9). From the third and fourth gels one can see that the colonies labelled as 1, 2, 3, 8, 9, 10 and 11 give the expected sized products. LB carbenicillin overnight cultures were grown from these seven colonies. The miniprepped DNA used for transforming these colonies was sent for sequencing.

Friday 24th August 2007

1. Maija miniprepped the seven Pr+XylR in TOPO cultures and run 1 µl of each plasmid on a gel. The gel showed weird size bands and we desiced to wait and see until we get the sequencing results from Germany.
2. Christine set up overnights for the following transformants, miniprepped them 25/08/07 and ran the minipreps on gel.

   Plate	Gene	Colonies	Expected size	Result
   1	phzA→phzG	A, B, C, D, E, F, G	10.2kb	No Insert
   2	phzA→phzD	A, B, C, D, E, F, G	6.7kb	No Insert
   3	phzD→phzG	A, B, C, D, E, F, G	7.4kb	Possible insert in B, C and D

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