Thursday, 2 August 2007

Research: Lab Techniques

Hi all, week 6 is here and it is my turn to share my SIP experience. Time flies, doesn’t it?

I’ve been assigned to a research lab for my SIP, so the scope of my duties is to basically carry out my MP which revolves around mbio/mgen.

My MP in a nutshell is to study protein interactions in two different gene constructs using a yeast-two hybrid system. This is to be used as a basis for further studies by the company to determine if protein interactions are involved in the activation of nucleus translocation signal in the 2 genes I am studying (please don’t ask me how, I wish I knew how they would study it, but a student like me isn’t privy to such info).

Anyways, gene construct refers to a vector that has been ligated with an insert of interest. I need to amplify the inserts (2 types) and ligate them into a vector which confers kanamycin resistance for selection upon plating.

This week I shall focus on the first part of my MP – constructing fusion genes. For those interested, I will talk about the yeast-two-hybrid in a later posting as it is quite a bit of theory

Constructing fusion genes:

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

1. Amplifying insert:
The first step is to obtain the inserts 1 and 2 by PCR (polymerase chain reaction) from a template DNA, provided in stock by the company. The underlying principle of PCR is that a single dna is all that is needed to generate many copies of replicate DNA.
There are 5 steps in total:
1) initial denaturation – 95oC, 4mins
2) denaturation - 95oC, 30s
3) Annealing - 56oC, 30s
4) Extension - 72oC, 4mins
5) Final extension - 72oC, 10mins
Step 2-4 run for 35 cycles.

The first denaturation is to separate dDNA so that the single strands can act as a template for synthesis. Annealing temperature is for the primers to anneal to the template and extension is for the polymerase to incorporate dNTPs for synthesis. I use PFU polymerase as it has proof reading ability – which is necessary to avoid errors in amplified sequence. Quality of PCR products can be affected by MgCl2 concentration, annealing temperature (too high no products, too low unspecific), genomic contaminations, amount of template DNA (I find 2ul produces good enough results for me) etc. Master mix has to be made up - not provided. Mine is as follows:

1) pfu buffer - 5ul
2) pfu polymerase - 0.5ul
3) dNTP -1ul
4) water - make up to 50ul reaction
5) Primers (forward and reverse) - 0.5uL each
6) Template (2ul)

Note: It is best to keep pfu polymerase on ice before placing to PCR to enhance its enzyme activity as it is not as heat stable as taq polymerase.

To view PCR products, need to run on agarose gel. I usually make up 1% agarose (agarose powder and TAE buffer) due to my insert size - 1=185bp, 2=1.8kbp and run for about 40mins at 100V, depending on size of the gel. Larger agarose concentrations (1.5-2%) are generally better for smaller dna chains <100bp.

As seen in the gel picture, there are some unspecific products – probably due to low annealing temperature. I did try raising the temperature by 1oC, but it produced no bands. My PCR products are the 200bp and 2kbp bands. These bands are excised under UV light using disposable scalpels and placed into 1.5mL microcentrifuge tubes for purification to send for sequencing. As a safety precaution, it is necessary to wear protective headgear before operating the UV illuminator.

Purification/gel clean up system: Wizard SV Gel and PCR clean-up system

After obtaining the bands, need to weigh them to determine how much membrane binding solution is needed.10ul/10mg of membrane binding solution (MBS) is utilsed, and gel excised is incubated with MBS at 50-65 oC till gel is completely dissolved. Mixture is transferred to minicolumn assembly (minicolumn + collection tube) and incubated at room temperature for 1 min, before centrifuging at 16000g for 1 min. This is to get rid of waste. Flow through is discarded.
700ul of membrane washing solution is added, and tubes spun at 16000g for 1 min, flowthrough discarded. Membrane washing solution (MWS) contains ethanol to precipitate DNA, otherwise DNA will be washed away. Washing step is repeated with 500ul of MWS and spun for 5mins. Tubes are then spun for 1 min to allow for evaporation of ethanol, as ethanol contamination with DNA can cause problems in sequencing and PCR reactions.
Lastly, water is added to elucidate purified DNA in column and centrifuged down for 1min. A portion of DNA obtained will then prepared for sequencing.

2. Sequencing:
Although we send sequencing to external companies, we need to prepare and perform the sequencing reactions ourselves. We are provided with a sequencing master mix called big dye which basically contains buffer, labeled ddNTPs etc. Set up is as follows:

Big dye -8ul
Water – 9.5ul
Template (DNA to be sequenced) – 2ul
*primer – 0.5ul
Total: 20ul

PCR conditions:
1) initial denaturation – 95oC, 3mins
2) denaturation - 95oC, 30s
3) Annealing - 56oC, 30s
4) Extension - 72oC, 4mins
5) Final extension – N/A
2-4 run for 30 cycles.

*Note that forward and reverse primers are added into 2 separate tubes. For every 1 template, there are 2 tubes – 1 forward and 1 reverse. This is for counterchecking purposes in sequencing. Lets say that the forward sequence result has 1 base pair discrepancy. To check if this is a mutant or if the sequencing was read wrongly by machine (higher chances if machine reports low confidence for that nucleotide, which can be quite random due to weak signaling of labeled fluorescent), it is counterchecked against the reverse sequence (need to reverse complement it first). If the sequence is as expected, then the error was due to error in reading. If the sequence produced is identical to the forward sequence, then the gene is probably mutated – the polymerase incorporated a wrong base pair.

Below is an example of sequencing results. The circled blocks are representation of the signal strength – the stronger the signal strength, the more sure we can be that the nucleotide is read correctly by the machine. Blue is the strongest (above 50%), yellow indicates a 40% chance and anything below 30% is red. Software used is sequence scanner from applied biosystems.

To check if the insert is correctly sequenced as expected – compare actual and expected sequence using NCBI Blast program:> blast>bl2seq>wblast2.cgi
If result is a 100% match, then it is considered correct. Anything lesser requires either re-sequencing or checking with reverse complement as mentioned previously (produce a forward sequence from a reverse primer). Reverse sequences can be reverse complemented manually or by using this program:>sms>rev_comp.html

3. Digestion:

Once it is confirmed that the sequences for both insert 1 and 2 are correct, they can be digested for ligation with the vector. The vector also needs to be digested.

Restriction enzymes such as EcoRI and NdeI are used. Since I do double digestion (add both enzymes at once), a compatible buffer for both must be used, such as buffer 4 from biolabs. Digested products are incubated at 37 oC (vector = 2hrs), (inserts = 4hrs).

Digested products are heated at 65 oC, 10mins to deactivate enzymes.
Digested vectors must be run on gel to obtain digested vector (non-digested vectors appear as smudge on gel. Gel picture below shows unsuccessful digestion (1) and successful digestion (2) of vector (ignore the other products).

Vector will then be excised and purified as described earlier, then dephosphorylated to prevent re-ligation. Alkaline phosphatase only works with in alkaline conditions (hence the name), so buffer is added.

Alkaline phosphatase – 5ul
AP buffer – 5ul
Vector - 40ul

Incubate at 37 oC, 1 hr, heat inactivate at 65oC, 30mins.

4. Ligation:
Here comes the easiest part by far! Basically, inserts are added to vectors (ratio 3:1 for higher success of ligation), and ligase and ligase buffer (I use T4) is added to seal the “nicks”. For those who can’t recall, nicks are the missing phosphate backbone. Incubate at 16 oC overnight for best results.

Ligated products will then be transformed in ecoli cells via heat shock to screen for successful colonies, which is step 5 -7 of the whole procedure - I will discuss this in my next posting as it seems a bit overkill now.

Sorry for the long post! Luckily, this stuff is all sem 2 work, so won’t be tested! Feel free to ask questions.


Debra , TG02


Star team said...

Hey Debra
Before you perform purification, you excised the gel. How do you excise the gel? Is it done mannually or automated? See u =)

Eugene Wong

VASTYJ said...

Hi Debra!

From the post, u mention that
'Larger agarose concentrations (1.5-2%) are generally better for smaller dna chains'.

May i know the reason for this? issit bcoz the agarose will 'trap' the dna better

Chaur Lee

VASTYJ said...

hey debra,

Why is it that digested vectors are incubated at 37oC for 2 hrs and digested inserts for 4 hours?

Andre, TG01

J.A.M.M.Y.S said...

Hey Debra

Just to let you know, my group have had the opporutnity to view first-hand how DNA sequncing is done. If you want/need to know about it more you can always ask either me or my other group members.

Anyways I have only one question to ask, my group used this free downloadable program called FinchTV to view the sequencing results, may I know wad program you are using?

Azhar TG01

Anonymous said...

Hi, I got 1 question. How do u select for kanamycin resistence?

Yeng Ting

MedBankers said...

Ah, i wish it was automated =(. We excise the gel manually, under UV light. Remember mbio? exactly like the practical. Bands will fluoresence green under UV, so simply choose your band of interest and use a scapel to cut it out, then put it to a 1.5ml tube for purification.
Hope that is clear enough.

Chaur Lee:
Yeap, you more or less got it. Agarose is from seaweed, and it forms a mesh like structure in the gel, which impedes the DNA. So, smaller DNA will be able to move through with more ease - faster, than larger fragments. Hence if you're trying to get smaller bands, it'll be better to use higher concetrations so that they don't run off the gel. Whereas if you want to get larger bands, a lower % of agarose is better as it allows a larger degree of seperation. Hope this ans your question.

Ah, this is part of my trouble shooting. See, there is this sequence in my vector that is a few bp different from the EcoRI restriction site. So if i digest for too long, the EcoRI gives start activity and actually overdigests (digest the similar, but non-specific site). Whereas there is no such sequence in the insert and can be digested for longer. Also, the inserts retriction sites are manually inserted into the primers, so it needs a longer time to digest.

MedBankers said...

Wow, how i envy you - that sounds pretty interesting.

I do have finchTV as well, but i choose to use seqeunce scanner by applied biosystems as it is more idiot-proof. :)

Personally, I took 1 look at finchTV and got a bit put off. It kinda gives nonsense when it can't determines the basepair, dont you think?

Yeng Ting:
well, the vector has kanmycin gene that confers for kanamycin resistance. So basically, to select for successful clones, cimply plate onto kanamycin plates. Those that contain the vector will grow on the plates as they have kanamycin resistance. Unsuccessful cells will not grow as the antibiotic would have impeded their growth.

Hope that ans all of your questions!

- Debra

royal physicians said...

hello~ I finally understood why u have to keep repeating ur experiments!! so tough =P haha.


Anonymous said...

haha...yeah. redoing that for the past 6 weeks! T_T.

getting the inserts is easy. optimising the digestion is horrible.

Hows your project coming along? Oh lunch time is flexible, so if you wanna eat, just give me a call yeah? will join you if not running exp at that time =)

- Debra

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