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Thanks everyone for your patience and attention.

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Thanks everyone for your patience and attention.

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I really enjoyed myself with you all. It's time for me to 'mug' with your reports. Yawn...

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It's time to start compiling your report and submitting it.

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It's time to start compiling your report and submitting it.

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Hi Johann! All groups had two bands for our SDS PAGE positive control 2... since its consistent across all groups, it cant be human error right? plus, our pro aka you did it... so was there sth wrong with the control itself? :O

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Your friend says he misses you.

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Hi all,

Thank you for those who came today. Despite some awkwardness, we pretty much gotten some things done. Good luck for your report writing.

I think I will still be available on Wednesday (12/4) between 1pm - 3pm. I know it is very close to the deadline, but I'll be there in case you need to urgently confirm something before you compile and submit it online. If not, we can always have a chat.

I'll see you then.

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For the buffer prep, since the amount of Sodium Phosphate and Sodium Chloride are the same throughout the three buffers, can we just put a statement saying they remain the same?
Or can we use a table to record the amounts, but show the formulas of how to calculate them at one side?
Thanks! :)

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For those who are interested what Fc region stands for - fragment crystallizable region.

But you don't need to use the full jargon; Fc region is sufficient.

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I did a rough sketch of your groups' purification profile plots. As you can see, everyone's profile is similar. As you move along the fractions, the S.E.A. increases drastically. yet, the concentration dips as well. Do you see a difference between the NI and I fractions?

As mentioned, IF you can use this plot to improve the way you write your report, by all means do so. IF you DO NOT understand this at all, I strongly suggest that you try not to attempt using it. Just focus on the purification table results instead.

*Plot sequence from top to bottom: Group 47, 48 & 49*

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Guys, tomorrow is the last practical of the semester. Happy?

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Guys, tomorrow is the last practical of the semester. Happy?

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So fast right?

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Some things to consider while writing your second report

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Some things to consider while writing your second report

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Hi all,

Please bear in mind that all of these questions are meant to be supplementary to your report. I.e., if you already feel that you have sufficient points (after going through the checklist in the guidelines), then it is not necessary to use these pointers below:

·         Is there any difference between the non-induced and induced cells?

o   Purification table parameters

o   Identification of possible LDH band from its size (compare with theoretical MW) on both gel and membrane.

o   Obvious thickening of the LDH band (on gel and membrane) after induction.

·         Is there any relationship between the parameters (of the purification table) and the samples?

·         What is the molecular weight of LDHA subunit? Do you observe a band of similar MW in the other fractions as to the positive controls? How about the elute fraction?

·         How is the purity of the elute fraction (single major band or presence of other bands)? If there are other bands, what can you say about the current purification technique? Is there any way to improve the system (if you are intending to do this, please give known scientific examples to substantiate your claims)?

o   Hint: Purification factor and yield.

·         Do you observe a correlation between the intensity of the LDH band and the specific enzyme activity/purification factor/yield? How do you compare it with the protein assay data?

·         What’s the reason for observing a single band in western blot vs. multiple bands in SDS-PAGE?

·         Is there a difference in intensity of the LDH bands in western blot as compared to SDS-PAGE, especially in the crude extracts? If so, is there any reason to the difference?

·         Do you observe bands in both of your positive controls? Why yes or no?

·         Is the correlation of the band intensities/thickness similar to the previous observed parameters?

·         Do you encounter any “problems” during your experimentation?

o   With protein quantification, enzyme assay data, etc.

o   With SDS-PAGE total protein staining.

o   With western blot data (non-specific bands, etc.)

Hope this will help you. Good luck in your report writing.

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So whatever you guys experienced once, I'm experiencing it at least three times more

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So whatever you guys experienced once, I'm experiencing it at least three times more

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Sigh...

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Thanks to Cheryl, Pei Xuan and Kai Chirng for your data. I have checked them, and the calculations seems fine. Other than group 49, please do furnish the units of the parameters by the time you submit your report.

I know at times I might sound sarcastic when we discuss about some things, but please do try to understand that I have no intention to do so. It can be frustrating to fight against time, to figure out what the terminologies/jargon mean. Yet, you must understand the basis of why we use these techniques for. And then have the ability to translate them into data values for explanation. Otherwise, it will be harder for you to write the report.

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1)      His SpinTrap (GE Healthcare, Singapore)

2)      Bio-Rad Protein Assay (Biorad, Singapore)

3)      InstantBlue Protein Stain (Expedeon, Singapore)

4)      Trans-Blot Turbo Transfer System (Biorad, California, USA)

5)      Anti-6X His tag antibody (Abcam, Cambridgeshire, England)

6)      Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP (ThemoFisher Scientific, Waltham, USA)

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I apologize if I was not able to be there for everyone yesterday. Practical 7 may have similar experience.

I would want you all to start drafting your reports so that you won't have to rush at the end. The calculations in Practical 6 can be daunting, but take some time to understand the difference between those terms. A lot of students cannot accept the idea between amount and concentration; there is a large difference between them. Another problem students have issues with is the volume (of assessment) in each of the fractions. Many cannot understand why Crude After Induction (aka Induced condition) should be 600uL instead of 2000uL. It is because only a portion of extract/lysate (600uL) was introduced into the column. So the assessment is only based on that volume.

Just in case some of you are wondering - you don't need to calculate the total protein amount, total enzyme activity, purification factor and yield for Crude Before Induction (aka Non-Induced cells). This is because this do not contribute to the column at all.

Consider the difference between purification factor and yield as well. The Induced cells will always have a purification factor of 1 and yield of 100%. I would want you to think about their relationships - what does it mean to have the set of values that you have?

Please do pass me your enzyme kinetics data whenever possible. I would want to study your values before we hit practical 8. Last but not least, get some idea on how to cast PAGE gels ya? We will be doing that next week. Good luck.

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Hi all, I just want to let you know that it has always been tradition that students will only get back their first report during the last practical. This is usually because of all of the vetting and counter-checks. We want to make sure that no one is credited wrongly ya? And that is why it takes time.

But none of the content in the first report will be useful for your second. The ONLY thing you probably can adapt is the writing style/formatting. So don't bother about the first report for now; focus on the second one. Collate your data and start making the graphs/figures/tables.

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I haven't read all of the reports yet, but I was hoping that someone would have displayed the colony numbers like this.

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I haven't read all of the reports yet, but I was hoping that someone would have displayed the colony numbers like this.

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It's ok. Either way is also acceptable.

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Hi all,

Tomorrow's consultation will be from 10am - 3pm instead because I have something urgent to attend to. I'm sorry. The venue is still the same; ls lab 8.

See you tomorrow.

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Ok guys, you ready for Practical 5 tomorrow?! We'll be moving at a faster pace ya?

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Ok guys, you ready for Practical 5 tomorrow?! We'll be moving at a faster pace ya?

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can I just confirm if our agarose gel had EtBr inside, or only SYBR safe, or both? Thanks

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Hi all, today is the end of your CA and you'll be wrapping up your report before Monday. What I need you to do is to start getting familiar with some protein techniques. PAGE casting will be one of them. So I would want you all to take a look at this video (https://youtu.be/5S4GJaFisJ8) to familiarize with the casting technique. We will discuss briefly on the technique during practical 5. For those who already had experience in it, I would suggest that your peers get to try it instead (unless all 4 of you already tried it before).

Thanks and see you next Wednesday

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Hiiii Dean here. For the lab report M&M dna sequencing portion, do we need to include the procedures after step 4 (thermocycling) since it was sent externally?

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Hiiii Dean here. For the lab report M&M dna sequencing portion, do we need to include the procedures after step 4 (thermocycling) since it was sent externally?

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Thanks!!

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Hi all, you can use this table as a guide but if you have a better way of organizing your data, by all means go ahead.

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Gentle reminder of this Fri's (24/2) consultation. We'll have it in ls lab 8 from 9am - 3pm ok? I need to rush of to somewhere else after that

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I have checked:

Group 47 - Good sequencing results. Got things to write. So far, based on my analysis, I saw 1 true and 2 sets of fake mutations.

*Updates*

Group 48 - Very good sequencing results. Your sequences are a little tricky, so be careful. But in my analysis, you have no mutations at all.

*Updates*

Group 49 - Good sequencing results as well. I see 1 true and 3 sets of fake mutations.

So good luck everyone.

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When you guys gotten your respective sequences, you will need to do four main things:

1) Identify each sequence; is it the mouse LDH A gene that we are looking.

2) Combine respective sequences to form contigs.

3) Compare contigs with the accession no. and switch the CDS feature on.

4) Identify fake or true mutations by comparing the graphic overview and the actual hit results.

*Updates*
In the appendices, you will need:
1) standard carves of P1B and P3
2) Hit results of individual sequence result to signify that it is the Mm LDHA gene
3) both F & R contigs with the features annotated
4) hit results (with the CDS features) of the F & R contigs vs accession no; identify where the mutations (if any) fall

They will constitute 11 pages altogether.

In the results, you will need:

1)  the overview graphic snapshot of the F & R contigs vs accession no.
2) a tabulated list of all of the mutations (be it fake or real mutations)

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It may be a little blur, but I hope you can see. We came up with a rough algorithm for it. So far we haven't seen it fail. But if you consider the XbaI scenario, we are able to get 987 fragments as well. So hope this will help you in future similar questions. Of course, the best way is to go back to conventional methods. Will take a long time though.

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Determining orientation from T-A or blunt end cloning

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Determining orientation from T-A or blunt end cloning

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The arrows are the orientation of protein transcription should be. You need to choose one cut in the vector and one in the insert (closest to either end; as long as it is not in the middle) to determine the orientation.

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You are using a XbaI to cut an E. coli genomic DNA (4,600,000 bp), and XbaI is sensitive to Dam methylated sites. How many fragments will you get possibly get? *remember how you got for ClaI restriction enzyme*

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Just a re-cap - We have no significant difference between all three independent experiments. We can then say that based on the experiments, using a 1:2 vector:insert ratio is considered counter-intuitive. 1:1 ratio would be considered an optimal level to get the most colonies. But at the same time, in statistics, it is perceived as insufficient data to conclude our findings. In the sense, it is in the assumption (from the beginning) that we thought both ratios SHOULD be different. So to prove this null hypothesis wrong, we would need more information (as in data) to state otherwise under a 95% confidence interval. Alternatively, we can reduce the confidence interval to maybe 75% or lesser to get what we want, but that's a cheap way of doing things.

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Everyone busy at work. Pract 3 already guys

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Everyone busy at work. Pract 3 already guys

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Alright guys (and gals), you have already seen each others' colony numbers. What you can do is to have vector:insert ratios 1:1 as one group, while 1:2 as the other. Use a statistical program that you are familiar with to check for significant difference.

The simpliest one I can think of is this webapp - https://www.graphpad.com/quickcalcs/ttest1.cfm

Key in the details for a student t-, two-tailed test.

Then ask yourself - if it is significantly different, what does it mean; if it is not sign. diff. then what can we say? We can discuss this tomorrow.

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Group 49 colony count after transformation

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Group 49 colony count after transformation

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#1 - 1992
#2 - 1064
#3 - 1488
#4 - 2432

(1:1) - #1 and #3 - total 3480
(1:2) - #2 and #4 - total 3496

- Eileen

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(1:1) plate 1- 67
(1:1) plate 2- 47
Total = 114
(1:2) plate 1- 23
(1:2) plate 2- 20
Total = 43

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(1:1) 572 (plate1) + 184 (plate 2)=756
(1:2) 103(plate 3) + 56 (plate 4)= 159

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If you have any doubts/questions, please post it up here ya?

You can also use the "attach file" function to take photos of your drawings if necessary.

You can also use your mobile as an alternative.

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Kits that we have used in Molecular part

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Kits that we have used in Molecular part

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TRIzol reagent (Invitrogen, Singapore) – Practical 1

First Strand cDNA sSynthesis Kit (ThermoFisher Scientific, Waltham, USA) – Practical 1

FavorPrep PCR Clean-up Kit (Favorgen, Taiwan) – Practical 1 & 2

DreamTaq DNA Polymerase (ThermoFisher Scientific, Singapore) – Practical

T4 DNA Ligase (Promega, Madison, USA) – Practical 2

NdeI (NEB, Ipswich, USA) – Practical 1 & 3

BamHI (NEB, Ipswich, USA) – Practical 1 & 3

EcoRI (NEB, Ipswich, USA) – Practical 1 & 3

Wizard Plus SV minipreps DNA Purification Systems (Promega, Madison, USA) – Practical 3

BigDye Terminator v3.1 Cycle Sequencing Kit (ThermoFisher Scientific, Waltham, USA) – Practical 4