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PROJECT LAB BOOK

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Welcome to the Macquarie University project lab book page!

DAY-BY-DAY PROGRESS FOR THE TRANSFORMATION AND CLONING

22nd September 2010

Plasmid Prep using Qiagen Plasmid Midi Prep Kit

The pET-3A vector plasmid that is to be used for the transformation needs to be purified

The pET-3A plasmid was purified using the Qiagen Plasmid Midi Prep Kit as per the manufacturer’s protocols

The only difference made to the protocol is that only 5ml of Buffer QC was used on the 2nd wash step

The plasmid (two samples A & B) as well as some column flow-through collected at different stages of the purification were run on a GelRed post-stained 1.5% agarose gel and photo taken for visualization (see below).

A NanoDrop spectrophotometer reading was also recorded to check the quality of the extracted genomic DNA.

There was no DNA pellet formed at the end of this protocol so we may have lost some DNA

There was also some plasmid observed in some of the column flow through so some plasmid has been lost

Figure 10. Qiagen Plasmid Midi Prep results:

Figure 10. GelRed post-stained 1.5% agarose gel of pET-3A Plasmid purification. In lanes 1, 4, 5 and 15 there is a 1kb Molecular ladder. In lane 2 there is the Plasmid purification sample A and in lane 3 is the Plasmid purification sample B. In lanes 6 to 14 there are flow-throughs that were collected at different stages throughout the protocol. There is some DNA visible in these lanes so some of the DNA has been lost. Possibly genomic DNA they are quite high (didn’t move through the wells)

Nanodrop results:

Sample	Concentration	A260/280 ratio
A	36.6ng/uL	1.71
B	35.0ng/uL	1.73

Looking at these results it seems that we have approximately 100uL of 36.6ng/uL plasmid sample A and approximately 100uL of 35.0ng/uL plasmid sample B.

A restriction digest will confirm that it is plasmid DNA not genomic DNA.

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27th September 2010

Plasmid Prep using Qiagen Plasmid Midi Prep Kit (2nd attempt)

We attempted to purify the pET-3A vector again using the Qiagen Plasmid Midi Prep Kit as per the manufacturer’s protocols

The plasmid (two samples A & B) as well as some column flow-through collected at different stages of the purification were run on a GelRed post-stained 1.5% agarose gel and photo taken for visualization (see below).

Figure 11. Qiagen Plasmid Midi Prep results (2nd attempt)

Figure 11. GelRed post-stained 1.5% agarose gel of pET-3A Plasmid purification. In lanes 1 and 10 there is a 1kb Molecular ladder. In lanes 2-9 there is the pooled plasmid purification samples that have been collected in separate tubes. All samples show a band indicative of linearized plasmid except lane 7 – this sample has been mistreated in the purification process.

Figure 12. Qiagen Plasmid Midi Prep results (2nd attempt)

Figure 12. GelRed post-stained 1.5% agarose gel of pET-3A Plasmid flow through collections. In lanes 1 and 9 there is a 1kb Molecular ladder. In lanes 2 – 9 there are the column flow throughs that were collected at different points throughout the protocol. In lanes 2 and 3 there is a feint band visible but this is expected as it is sample collected before the addition of sample to the column.

Less DNA has been lost in the column flow through with this attempt however as we suspect that the plasmid is still contaminated with genomic DNA we decided to be safe and use another type of plasmid prep kit as well.

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31st September 2010

Plasmid Prep using Invitrogen Midi Prep Kit

The pET-3A plasmid was purified using the Invitrogen Midi Prep Kit as per the manufacturer’s protocols

The plasmid (two samples 1 & 2) were run on a GelRed post-stained 1.5% agarose gel and photo taken for visualization (see below).

A band was seen for sample 2 but no band was observed for sample 2

This protocol has been successful but not enough plasmid has been purified

Figure 13. Plasmid Prep using Invitrogen Midi Prep Kit results

Figure 13. GelRed post-stained 1.5% agarose gel of pET-3A Plasmid purification using Invitrogen Midi Prep kit. This kit shows that we have recovered less plasmid but it is more highly purified than what was purified with the other kits because the bands are less smeared.

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31st September 2010

Restriction enzyme digest on Purified pET-3A

The enzyme digest was performed on the pET-3A vector that had been purified with the Qiagen Midi Prep protocol (performed on 27th September) as well as the pET-3A vector that had been purified with the Invitrogen Midi Prep kit (performed on the 31st September)

The pET-3A vector was digested with BamH1 and Nde1 restriction enzymes (see recognition sites below) so that the D. radiodurans and A. tumefaciens bacteriophytochrome-RBS-HO site operons that we have constructed can be ligated into the vector

The digests were run on a GelRed stained 1% agarose gel and photo taken for visualization (see below).

The restriction digest confirmed that it is plasmid DNA that has been purified is linearized and not circular as it collapsed to a single band of expected size and intensity

Restriction enzyme recognition sites:

Restriction enzyme	Recognition site
BamH1	5’-CA (cut) TATG – 3’ and 3’-CCTAG (cut) G-5’
Nde1	5’-CA(cut)TATG-3’and 3’-GTAT(cut)AG-5’

The reaction mastermix for the restriction digest to be performed on the Qiagen Midi Prep purified plasmid was set up as per the following recipe (per sample):

Mastermix:	Amount per sample (uL)
Gibco H2O	7.5
10x Buffer	10.0
BSA (10ug/uL)	0.5
pET-3A DNA	1.0
Restriction enzyme	1.0
Total	20.0