Research: Lab Techniques

Hi all, we’re about 60% through with SIP! As promised, I shall continue where I left off from my previous post. Previously, I stopped at ligation. Here is a quick recap:

Steps:
1. Amplify gene 1 (PCR) and gene 2 (PCR)
2. Send amplified genes for sequencing (outsource to external company)
3. Digest vector, inserts – 1 and 2
4. Ligate inserts to vector
5. Transform ligated products
6. Colony PCR (screen for inserts in transformed cells)
7. Send positive clones from colony PCR for sequencing

I will now cover step 5.

5. Transformation
For my case, I use commercially competent TOP10 Ecoli cells, so I can use the heat shock method. Set up is as follows:

1) deactivate ligase by incubating at 65oC for 10 mins. This step is optional. Some even suggest that inactivating ligase may affect transformation efficacy. I have done both and for me there is no considerable difference.
2) Add 50ul of competent cells into 5-10ul of ligation mix. Adding too much may give rise to too many colonies, making it hard to isolate cells.
3) Ice mixture for 30 mins, before heat shock at 42oC for 2 mins. This is supposed to improve transformation efficiency.
4) Add 250ul of LB without antibiotics. Place on shaking incubator at 37oC for 1.5hrs. This is to allow the transformed cells to express the plasmid’s antibiotic resistance, in my case, kanamycin.
5) Spin down using microcentrifuge to obtain pellet. Resuspend in 100ml of LB.
6) Plate out on pre-warmed LB kan+ plates, and incubate overnight at 37oC.

Controls:
To check if transformation is working, a positive control is required. I use the original, circularized yeast vector (not digested) as a positive control. This however has its limitations. It merely tells me if my transformation process is working or not, not the efficiency I can expect. This is because cells take up supercoiled dna (smaller, vector appears as 5kbp on gel instead of it’s actual 7kbp due to it’s supercoiled state) much more easily than linearised plasmid which size has been made bigger due to the addition of the inserts (mine totals to 10kb and 8kb, and has been giving me problems). Hence sometimes I get good positive controls, but poor transformation on my recombinant cells.

Also, a negative control is necessary to ensure that kanamycin plates contain enough kanamycin concentration to prevent cells without resistance from growing – eliminate the possibility of false positive clones.

Lastly, a control using just the vector that has been double digested with restriction enzymes should be performed. There should be few or ideally no colonies present, as using 2 different enzymes will not produce sticky ends for self ligation. Colonies will indicate incomplete digestion by 1 enzyme, hence resulting in self ligation – 1 enzyme did not cut well, hence only 1 enzyme worked, producing sticky ends. To further prevent this, vector can be dephosphorylated. However, dephosphorylation can inhibit ligation and hence when 2 enzymes are used, dephosphorylation is not encouraged.

Kanamycin plates:
Usually made up with kanamycin concentration of 50ug/ul.
1) Warm up 400mL of LB + agar mixture using microwave at 30% power. The mixture heats up unevenly and has a tendency to boil over if not careful.
2) Allow mixture to cool at room temperatute in it’s liquid state.
3) Add 400ul of 50ug/ul kanamycin when mixture has cooled. This is so that the antibiotic will not be killed off by the heat. To be done in laminar flow hood to avoid possible contamination.
4) Mix bottle well, and pour onto empty plates. 400mL can make up 20 plates nicely. No specific amount needs to be poured, it is at user discretion. In other words, guesstimate. Best to keep as equal as possible though qualitatively, from plate to plate.
5) Allow plates to solidify in 37oC incubator. When solid, open cover to allow excess water due to condensation to evaporate, before storing in fridge for future use.

That’s about it for transformation process. Any queries, questions, comments don’t hesitate. I shall cover colony PCR and other techniques like restriction digestion and sequencing in the next update, week 18.

Till then,

Debra, TG02